KOC1-derived peptide and vaccine including same

ABSTRACT

The present invention provides KOC1-derived epitope peptides having the ability to induce cytotoxic T cells. The present invention further provides polynucleotides encoding the peptides, antigen-presenting cells presenting the peptides, and cytotoxic T cells targeting the peptides, as well as methods of inducing the antigen-presenting cells or CTLs. The present invention also provides compositions and pharmaceutical compositions containing them as an active ingredient. Further, the present invention provides methods of treating and/or preventing cancer, and/or preventing postoperative recurrence thereof, using the peptides, polynucleotides, antigen-presenting cells, cytotoxic T cells or pharmaceutical compositions of the present invention. Methods of inducing an immune response against cancer are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a § 371 National Phase Application ofPCT/JP2015/071829, filed Jul. 31, 2015, which application claimspriority to JP 2014-158919, filed Aug. 4, 2014, JP 2014-158920, filedAug. 4, 2014, and JP 2014-158921, filed Aug. 4, 2014, the disclosures ofwhich are hereby incorporated by reference in their entireties for allpurposes.

REFERENCE TO A SEQUENCE LISTING

The Substitute Sequence Listing written in fileSubstituteSequenceListing_087331-029800US-1036284.tx created on Apr. 19,2017, 39,504 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy. In particular, the presentinvention relates to novel peptides that are effective as cancervaccines, methods for either or both of treating and preventing tumorsusing the peptide(s), and pharmaceutical compositions comprising thepeptide(s).

The present application claims the benefit of Japanese patentapplications filed on Aug. 4, 2014 (Japanese Patent Application Nos.2014-158919, 2014-158920, and 2014-158921), the entire contents of whichare incorporated by reference herein.

BACKGROUND ART

Cytotoxic T lymphocytes (CTLs) have been shown to recognize epitopepeptides derived from the tumor-associated antigens (TAAs) found on themajor histocompatibility complex (MHC) class I molecule, and then killthe tumor cells. Since the discovery of the melanoma antigen (MAGE)family, many other TAAs have been discovered through immunologicalapproaches (NPL1: Boon T, Int J Cancer 1993 May 8, 54(2): 177-80; NPL2:Boon T & van der Bruggen P, J Exp Med 1996 Mar. 1, 183(3): 725-9). Someof these TAAs are currently undergoing clinical development asimmunotherapeutic targets.

In several of these TAAs, epitope peptides that can be recognized byCTLs are identified and their application in immunotherapy for varioustypes of cancer is anticipated (NPL3: Harris C C, J Natl Cancer Inst1996 Oct. 16, 88(20): 1442-55; NPL4: Butterfield L H et al., Cancer Res1999 Jul. 1, 59(13): 3134-42; NPL5: Vissers J L et al., Cancer Res 1999Nov. 1, 59(21): 5554-9; NPL6: van der Burg S H et al., J Immunol 1996May 1, 156(9) 3308-14; NPL7: Tanaka F et al., Cancer Res 1997 Oct. 15,57(20): 4465-8; NPL8: Fujie T et al., Int J Cancer 1999 Jan. 18, 80(2):169-72; NPL9: Kikuchi M et. al., Int J Cancer 1999 May 5, 81(3): 439-66;NPL10: Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94). Untilnow, several clinical trials using these TAA-derived CTL epitopepeptides have been reported. Unfortunately, many of these clinicaltrials show a low objective response rate (NPL11: Belli F et al., J ClinOncol 2002 Oct. 15, 20(20): 4169-80; NPL12: Coulie P G et al., ImmunolRev 2002 October, 188: 33-42; NPL13: Rosenberg S A et al., Nat Med 2004September, 10(9): 909-15). Therefore, there is still demand foridentification of novel CTL epitopes that can be used in cancerimmunotherapy.

KOC1 (insulin-like growth factor II mRNA binding protein 3 alsodescribed as IGF2BP3 or IMP-3; reference sequence: GeneBank AccessionNumber NM_006547.2 (SEQ ID NO: 109)) is identified as a geneup-regulated in lung cancer by gene expression profile analysis using agenome-wide cDNA microarray containing 23,040 genes (NPL14: Kikuchi T etal., Oncogene. 2003 Apr. 10; 22(14): 2192-205; PTL1: WO2004/031413). TheKOC1 expression is observed to be up-regulated specifically in tumorcells in over 90% of lung cancer patients, but it is not expressed inother normal important organs except the testis and placenta. Further,it is shown that cell proliferation in KOC1-expressing cancer cell linesis suppressed as a result of down-regulation of the KOC1 expression byRNA interference.

Recently, KOC1-derived HLA-A24-restricted CTL epitope peptides (PTL2:WO2006/090810; NPL15: Suda T et al., Cancer Sci. 2007 November; 98(11):1803-8) and HLA-A2-restricted CTL epitope peptides (PTL3: WO2011/067920;NPL16: Tomita Y et al., Cancer Sci. 2011 January; 102(1): 71-8) havebeen identified. These peptides are effective in cancer patients havingthe HLA-A24 type or HLA-A2 type, but cannot be expected to have effecton cancer patients who do not have these HLA types.

CITATION LIST Patent Literature

-   [PTL 1] WO2004/031413-   [PTL2] WO2006/090810-   [PTL3] WO2011/067920

Non Patent Literature

-   [NPL 1] Boon T, Int J Cancer 1993 May 8, 54(2): 177-80-   [NPL 2] Boon T & van der Bruggen P, J Exp Med 1996 Mar. 1, 183(3):    725-9-   [NPL 3] Harris C C, J Natl Cancer Inst 1996 Oct. 16, 88(20): 1442-55-   [NPL 4] Butterfield L H et al., Cancer Res 1999 Jul. 1, 59(13):    3134-42-   [NPL 5] Vissers J L et al., Cancer Res 1999 Nov. 1, 59(21): 5554-9-   [NPL 6] van der Burg S H et al., J Immunol 1996 May 1, 156(9):    3308-14-   [NPL 7] Tanaka F et al., Cancer Res 1997 Oct. 15, 57(20): 4465-8-   [NPL 8] Fujie T et al., Int J Cancer 1999 Jan. 18, 80(2): 169-72-   [NPL 9] Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66-   [NPL 10] Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94-   [NPL 11] Belli F et al., J Clin Oncol 2002 Oct. 15, 20(20): 4169-80-   [NPL 12] Coulie P G et al., Immunol Rev 2002 October, 188: 33-42-   [NPL 13] Rosenberg S A et al., Nat Med 2004 September, 10(9): 909-15-   [NPL 14] Kikuchi T et al., Oncogene. 2003 Apr. 10; 22(14): 2192-205-   [NPL 15] Suda T et al., Cancer Sci. 2007 November; 98(11): 1803-8-   [NPL 16] Tomita Y et al., Cancer Sci. 2011 January; 102(1): 71-8

SUMMARY OF THE INVENTION

The present invention relates to peptides that can induce CTLs specificto KOC1-expressing cells. When these peptides are presented onantigen-presenting cells (APCs) by the human leukocyte antigen (HLA),CTLs that show a specific cytotoxic activity against KOC1-expressingcells are induced. KOC1-derived peptides that have been identified sofar to have CTL-inducing ability (CTL inducibility) are eitherHLA-A2-restricted or HLA-A24-restricted peptides, and cannot induce CTLsagainst cells that do not express these HLAs. Therefore, conventionalpeptides are not suitable for performing immunotherapy in subjects thatdo not have these HLAs. HLA-A11 and HLA-A33 are alleles commonly seen inAsians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12), andHLA-A03 and HLA-A01 are alleles commonly seen in Caucasians (Cao et al.,Hum Immunol 2001; 62(9): 1009-30). It is desirable to administerHLA-A11-restricted peptides to HLA-A11-positive subjects,HLA-A33-restricted peptides to HLA-A33-positive subjects,HLA-A03-restricted peptides to HLA-A03-positive subjects, andHLA-A01-restricted peptides to HLA-A01-positive subjects. Hence, thepresent invention relates to KOC1-derived peptides with CTL-inducingability that are restrictive to HLA-A11, HLA-A33, HLA-A03, or HLA-A01.Based on results disclosed herein, the peptides of the present inventionhave been proven to be epitope peptides that can induce a potent andspecific immune response against cells expressing KOC1 and HLA-A11,HLA-A33, HLA-A03, or HLA-A01.

Therefore, one of the objectives of the present invention is to provideKOC1-derived peptides that can induce CTLs in an HLA-A11-, HLA-A33-,HLA-A03- or HLA-A01-restrictive manner. These peptides can be used toinduce CTLs in vitro, ex vivo or in vivo, or can be used to administerto subjects for the purpose of inducing an immune response againstKOC1-expressing cancer cells. Preferable peptides are peptidescomprising the amino acid sequence selected from among SEQ ID NOs: 5,28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90,92, 46, 95 and 41; more preferable peptides are nonapeptides ordecapeptides; and even more preferable peptides are peptides consistingof the amino acid sequence selected from among SEQ ID NOs: 5, 28, 30,32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46,95 and 41.

The peptides of the present invention encompass peptides in which one,two or more amino acid(s) is/are substituted, deleted, inserted and/oradded, as long as the resultant modified peptides retain theCTL-inducing ability of the original peptide.

The present invention further provides isolated polynucleotides encodingany one of the peptides of the present invention. Similar to thepeptides of the present invention, these polynucleotides can be used forinducing APCs with CTL-inducing ability, and can be administered tosubjects for inducing an immune response against KOC1-expressing cancercells.

The present invention also provides compositions comprising one or moretypes of peptides of the present invention, one or more types ofpolynucleotides encoding one or more types of peptides of the presentinvention, APCs of the present invention, exosomes presenting peptidesof the present invention, and/or CTLs of the present invention. Thecompositions of the present invention are preferably pharmaceuticalcompositions. The pharmaceutical compositions of the present inventioncan be used for treating and/or preventing cancer, as well as preventingpostoperative recurrence thereof. They can also be used for inducing animmune response against cancer. When administered to a subject, apeptide of the present invention is presented on the surface of an APC,and as a result CTLs targeting the peptide are induced. Therefore,another objective of the present invention is to provide compositionsfor inducing CTLs, wherein the compositions comprise one or more typesof peptides of the present invention, one or more types ofpolynucleotides encoding one or more types of peptides of the presentinvention, APCs of the present invention, and/or exosomes presentingpeptides of the present invention.

A further objective of the present invention is to provide methods ofinducing APCs having CTL-inducing ability, wherein the methods comprisea step of contacting one or more types of peptides of the presentinvention with an APC, or a step of introducing a polynucleotideencoding any one peptide of the present invention into an APC.

The present invention further provides a method of inducing CTLs,comprising a step of co-culturing a CD8-positive T cell with an APC thatpresents on its surface a complex of an HLA antigen and a peptide of thepresent invention, a step of co-culturing a CD8-positive T cell with anexosome that presents on its surface a complex of an HLA antigen and apeptide of the present invention, or a step of introducing into aCD8-positive T cell a vector comprising a polynucleotide encoding eachsubunit of a T cell receptor (TCR) capable of binding to a peptide ofthe present invention presented by an HLA antigen on a cell surface. Thepreferred HLA antigen in the present invention is HLA-A11, HLA-A33,HLA-A03 or HLA-A01.

A further objective of the present invention is to provide isolated APCsthat present on their surface a complex of an HLA antigen and a peptideof the present invention. The present invention further providesisolated CTLs targeting a peptide of the present invention. These APCsand CTLs can be used in immunotherapy for KOC1-expressing cancers. Inthe present invention, the cancer to be subjected to immunotherapy is,for example, a cancer present in patients who have a homozygote orheterozygote of HLA-A11, HLA-A33, HLA-A03 or HLA-A01. That is, thepresent invention provides immunotherapy for cancers expressing KOC1 andat least one HLA antigen selected from HLA-A11, HLA-A33, HLA-A03 andHLA-A01.

Another objective of the present invention is to provide methods ofinducing an immune response against cancer in a subject, wherein themethods comprise a step of administering to the subject a peptide(s) ofthe present invention, a polynucleotide(s) encoding the peptide(s), anAPC(s) of the present invention, an exosome(s) presenting a peptide(s)of the present invention, and/or a CTL(s) of the present invention.Another objective of the present invention is to provide methods oftreating and/or preventing cancer, as well as preventing postoperativerecurrence thereof in a subject, wherein the methods comprise a step ofadministering to the subject a peptide(s) of the present invention, apolynucleotide(s) encoding the peptide(s), an APC(s) of the presentinvention, an exosome(s) presenting a peptide(s) of the presentinvention, and/or a CTL(s) of the present invention.

In addition to the above, other objects and features of the presentinvention will become more fully apparent when the following detaileddescription is read in conjunction with the accompanying figures andexamples. However, it is to be understood that both the foregoingsummary of the present invention and the following detailed descriptionare of exemplified embodiments, and not restrictive of the presentinvention or other alternate embodiments of the present invention. Inparticular, while the present invention is described herein withreference to a number of specific embodiments, it will be appreciatedthat the description is illustrative of the present invention and is notconstructed as limiting of the present invention. Various modificationsand applications may occur to those who are skilled in the art, withoutdeparting from the spirit and the scope of the present invention, asdescribed by the appended claims. Likewise, other objects, features,benefits and advantages of the present invention will be apparent fromthis summary and certain embodiments described below, and will bereadily apparent to those skilled in the art. Such objects, features,benefits and advantages will be apparent from the above in conjunctionwith the accompanying examples, data, figures and all reasonableinferences to be drawn therefrom, alone or with consideration of thereferences incorporated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 consists of photos (a) to (e) showing results of an interferon(IFN)-gamma enzyme-linked immunospot (ELISPOT) assay in CTLs inducedusing peptides derived from KOC1. In comparison with the control, CTLsin Well #6 with KOC1-A11-9-415 (SEQ ID NO: 5) (a), Well #4 withKOC1-A11-10-414 (SEQ ID NO: 28) (b), Well #5 with KOC1-A11-10-204 (SEQID NO: 30) (c), and Well #8 with KOC1-A11-10-14 (SEQ ID NO: 32) (d)showed potent IFN-gamma production. In these photos, the square on thewells show that cells from the respective wells were propagated for theestablishment of CTL lines. In contrast, KOC1-A11-9-258 (SEQ ID NO: 1)(e) is shown as an example of typical negative data where there was nospecific IFN-gamma production. In the figure, “+” shows IFN-gammaproduction against target cells pulsed with an appropriate peptide; and“−” shows IFN-gamma production against target cells that have not beenpulsed with any peptides.

FIG. 2 consists of a series of line graphs (a) to (c) showing results ofan IFN-gamma enzyme-linked immunosorbent assay (ELISA), confirmingIFN-gamma production in CTL lines stimulated with KOC1-A11-9-415 (SEQ IDNO: 5) (a), KOC1-A11-10-204 (SEQ ID NO: 30) (b) or KOC1-A11-10-14 (SEQID NO: 32) (c). These results prove that the CTL lines established bystimulation with each of the peptides showed potent IFN-gamma productionin comparison with the control. In the figure, “+” shows IFN-gammaproduction against target cells pulsed with an appropriate peptide; and“−” shows IFN-gamma production against target cells that have not beenpulsed with any peptides. The R/S ratio indicates the ratio of the cellnumber of CTL line (Responder cells) and the cell number of target cells(Stimulator cells).

FIG. 3 consists of line graphs (a) and (b) showing IFN-gamma productionin CTL clones established by limiting dilution from CTL lines stimulatedwith KOC1-A11-9-415 (SEQ ID NO: 5) or KOC1-A11-10-14 (SEQ ID NO: 32).These results prove that the CTL clones established by stimulation witheach of the peptides showed potent IFN-gamma production in comparisonwith the control. In the figure, “+” shows IFN-gamma production againsttarget cells pulsed with an appropriate peptide; and “−” shows IFN-gammaproduction against target cells that have not been pulsed with anypeptides. The R/S ratio indicates the ratio of the cell number of CTLclone (Responder cells) and the cell number of target cells (Stimulatorcells).

FIG. 4 is a line graph showing specific CTL activity against targetcells expressing both KOC1 and HLA-A*1101. COS7 cells transfected witheither HLA-A*1101 or the full-length KOC1 gene were prepared as thecontrol. The CTL clone established using KOC1-A11-10-14 (SEQ ID NO: 32)demonstrated a specific CTL activity against COS7 cells transfected withboth KOC1 and HLA-A*1101 (black diamond). On the other hand, asignificant specific CTL activity was not shown against target cellstransfected with either one of HLA-A*1101 (white triangle) and KOC1(white circle).

FIG. 5 consists of photos (a) to (l) showing results of an IFN-gammaELISPOT assay in CTLs induced using peptides derived from KOC1. Incomparison with the control, CTLs in Well #1 with KOC1-A33-9-543 (SEQ IDNO: 61) (a), Well #2 with KOC1-A33-9-282 (SEQ ID NO: 62) (b), Well #3with KOC1-A33-9-317 (SEQ ID NO: 63) (c), Well #8 with KOC1-A33-9-485(SEQ ID NO: 64) (d), Well #3 with KOC1-A33-9-286 (SEQ ID NO: 67) (e),Well #8 with KOC1-A33-9-34 (SEQ ID NO: 74) (f), Well #4 withKOC1-A33-10-542 (SEQ ID NO: 77) (g), Well #3 with KOC1-A33-10-281 (SEQID NO: 52) (h), Well #6 with KOC1-A33-10-543 (SEQ ID NO: 79) (i), Well#1 with KOC1-A33-10-424 (SEQ ID NO: 80) (j), and Well #5 withKOC1-A33-10-285 (SEQ ID NO: 85) (k) showed potent IFN-gamma production.In these photos, the square on the wells show that cells from therespective wells were propagated for the establishment of CTL lines. Incontrast, KOC1-A33-9-517 (SEQ ID NO: 4) (l) is shown as an example oftypical negative data where there was no specific IFN-gamma production.In the figure, “+” shows IFN-gamma production against target cellspulsed with an appropriate peptide; and “−” shows IFN-gamma productionagainst target cells that have not been pulsed with any peptides.

FIG. 6 consists of a series of line graphs (a) to (f) showing results ofIFN-gamma ELISA, confirming IFN-gamma production in CTL lines stimulatedwith KOC1-A33-9-543 (SEQ ID NO: 61) (a), KOC1-A33-9-282 (SEQ ID NO: 62)(b), KOC1-A33-9-485 (SEQ ID NO: 64) (c), KOC1-A33-9-286 (SEQ ID NO: 67)(d), KOC1-A33-10-542 (SEQ ID NO: 77) (e), or KOC1-A33-10-281 (SEQ ID NO:52) (f). These results prove that the CTL lines established bystimulation with each of the peptides showed potent IFN-gamma productionin comparison with the control. In the figure, “+” shows IFN-gammaproduction against target cells pulsed with an appropriate peptide; and“−” shows IFN-gamma production against target cells that have not beenpulsed with any peptides. The R/S ratio indicates the ratio of the cellnumber of CTL line (Responder cells) and the cell number of target cells(Stimulator cells).

FIG. 7 consists of a series of line graphs (a) to (f) showing IFN-gammaproduction in CTL clones established by limiting dilution from CTL linesstimulated with KOC1-A33-9-543 (SEQ ID NO: 61) (a), KOC1-A33-9-282 (SEQID NO: 62) (b), KOC1-A33-9-485 (SEQ ID NO: 64) (c), KOC1-A33-9-286 (SEQID NO: 67) (d), KOC1-A33-10-542 (SEQ ID NO: 77) (e), or KOC1-A33-10-281(SEQ ID NO: 52) (f). These results prove that the CTL clones establishedby stimulation with each of the peptides showed potent IFN-gammaproduction in comparison with the control. In the figure, “+” showsIFN-gamma production against target cells pulsed with an appropriatepeptide; and “−” shows IFN-gamma production against target cells thathave not been pulsed with any peptides. The R/S ratio indicates theratio of the cell number of CTL clone (Responder cells) and the cellnumber of target cells (Stimulator cells).

FIG. 8 consists of a series of line graphs (a) to (c) showing specificCTL activity against target cells expressing both KOC1 and HLA-A*3303.COS7 cells transfected with either HLA-A*3303 or the full-length KOC1gene were prepared as the control. The CTL clones established usingKOC1-A33-9-485 (SEQ ID NO: 64) (a), KOC1-A33-9-286 (SEQ ID NO: 67) (b),and KOC1-A33-10-542 (SEQ ID NO: 77) (c) demonstrated a specific CTLactivity against COS7 cells transfected with both KOC1 and HLA-A*3303(black diamond). On the other hand, a significant specific CTL activitywas not shown against target cells transfected with either one ofHLA-A*3303 (white triangle) and KOC1 (white circle).

FIG. 9 consists of photos (a) to (d) showing results of an IFN-gammaELISPOT assay in CTLs induced using peptides derived from KOC1. Incomparison with the control, CTLs in Well #5 with KOC1-A03-10-120 (SEQID NO: 27) (a), Well #3 with KOC1-A03-10-204 (SEQ ID NO: 30) (b), andWell #5 with KOC1-A03-10-281 (SEQ ID NO: 52) (c) showed potent IFN-gammaproduction. In these photos, the square on the wells show that cellsfrom the respective wells were propagated for the establishment of CTLlines. In contrast, KOC1-A03-10-414 (SEQ ID NO: 28) (d) is shown as anexample of typical negative data where there was no specific IFN-gammaproduction. In the figure, “+” shows IFN-gamma production against targetcells pulsed with an appropriate peptide; and “−” shows IFN-gammaproduction against target cells that have not been pulsed with anypeptides.

FIG. 10 is a line graph showing results of IFN-gamma ELISA, confirmingIFN-gamma production in a CTL line stimulated with KOC1-A03-10-120 (SEQID NO: 27). These results prove that the CTL line established bystimulation with each of the peptides showed potent IFN-gamma productionin comparison with the control. In the figure, “+” shows IFN-gammaproduction against target cells pulsed with an appropriate peptide; and“−” shows IFN-gamma production against target cells that have not beenpulsed with any peptides. The R/S ratio indicates the ratio of the cellnumber of the CTL line (Responder cells) and the cell number of thetarget cells (Stimulator cells).

FIG. 11 is a line graph showing IFN-gamma production in a CTL cloneestablished by limiting dilution from the CTL line stimulated withKOC1-A03-10-120 (SEQ ID NO: 27). These results prove that the CTL cloneestablished by stimulation with each of the peptides showed potentIFN-gamma production in comparison with the control. In the figure, “+”shows IFN-gamma production against target cells pulsed with anappropriate peptide; and “−” shows IFN-gamma production against targetcells that have not been pulsed with any peptides. The R/S ratioindicates the ratio of the cell number of the CTL clone (Respondercells) and the cell number of the target cells (Stimulator cells).

FIG. 12 is a line graph showing specific CTL activity against targetcells expressing both KOC1 and HLA-A*0301. COS7 cells transfected witheither HLA-A*0301 or the full-length KOC1 gene were prepared as thecontrol. The CTL clone established using KOC1-A03-10-120 (SEQ ID NO: 27)demonstrated a specific CTL activity against COS7 cells transfected withboth KOC1 and HLA-A*0301 (black diamond). On the other hand, asignificant specific CTL activity was not shown against target cellstransfected with either one of HLA-A*0301 (white triangle) and KOC1(white circle).

FIG. 13 consists of photos (a) to (h) showing results of an IFN-gammaELISPOT assay in CTLs induced using peptides derived from KOC1. Incomparison with the control, CTLs in Well #2 with KOC1-A01-9-96 (SEQ IDNO: 86) (a), Well #4 with KOC1-A01-9-118 (SEQ ID NO: 87) (b), Well #3with KOC1-A01-9-404 (SEQ ID NO: 90) (c), Well #4 with KOC1-A01-9-402(SEQ ID NO: 92) (d), Well 7# with KOC1-A01-10-312 (SEQ ID NO: 46) (e),Well #1 with KOC1-A01-10-402 (SEQ ID NO: 95) (f), and Well #1 withKOC1-A01-10-535 (SEQ ID NO: 41) (g) showed potent IFN-gamma production.In these photos, the square on the wells show that cells from therespective wells were propagated for establishment of CTL lines. Incontrast, KOC1-A01-9-536 (SEQ ID NO: 13) (h) is shown as an example oftypical negative data where there was no specific IFN-gamma production.In the figure, “+” shows IFN-gamma production against target cellspulsed with an appropriate peptide; and “−” shows IFN-gamma productionagainst target cells that have not been pulsed with any peptides.

FIG. 14 consists of a series of line graphs (a) and (b) showing resultsof IFN-gamma ELISA, confirming IFN-gamma production in CTL linesstimulated with KOC1-A01-9-96 (SEQ ID NO: 86) (a) or KOC1-A01-9-118 (SEQID NO: 87) (b). These results prove that the CTL lines established bystimulation with each of the peptides showed potent IFN-gamma productionin comparison with the control. In the figure, “+” shows IFN-gammaproduction against target cells pulsed with an appropriate peptide; and“−” shows IFN-gamma production against target cells that have not beenpulsed with any peptides. The R/S ratio indicates the ratio of the cellnumber of CTL line (Responder cells) and the cell number of target cells(Stimulator cells).

FIG. 15 is a line graph showing IFN-gamma production in a CTL cloneestablished by limiting dilution from the CTL line stimulated withKOC1-A01-9-96 (SEQ ID NO: 86). These results prove that the CTL cloneestablished by stimulation with each of the peptides showed potentIFN-gamma production in comparison with the control. In the figure, “+”shows IFN-gamma production against target cells pulsed with anappropriate peptide; and “−” shows IFN-gamma production against targetcells that have not been pulsed with any peptides. The R/S ratioindicates the ratio of the cell number of the CTL clone (Respondercells) and the cell number of the target cells (Stimulator cells).

FIG. 16 is a line graph showing specific CTL activity against targetcells expressing both KOC1 and HLA-A*0101. COS7 cells transfected witheither HLA-A*0101 or the full-length KOC1 gene were prepared as thecontrol. The CTL clone established using KOC1-A01-9-96 (SEQ ID NO: 86)demonstrated a specific CTL activity against COS7 cells transfected withboth KOC1 and HLA-A*0101 (black diamond). On the other hand, asignificant specific CTL activity was not shown against target cellstransfected with either one of HLA-A*0101 (white triangle) and KOC1(white circle).

MODE FOR CARRYING OUT THE INVENTION Description of Embodiments

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. However, before the present materials and methods aredescribed, it is to be understood that the present invention is notlimited to the particular sizes, shapes, dimensions, materials,methodologies, protocols, etc. described herein, as these may vary inaccordance with routine experimentation and optimization. It is also tobe understood that the terminology used in the description is for thepurpose of describing the particular versions or embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

I. Definitions

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

The terms “isolated” and “purified” used in relation with a substance(for example, peptide, antibody, polynucleotide or such) indicate thatthe substance does not substantially contain at least one substance thatmay else be included in a natural source. Thus, an isolated or purifiedpeptide refers to a peptide that does not substantially contain anothercellular material, for example, carbohydrate, lipid and othercontaminating proteins from the cell or tissue source from which thepeptide is derived. When the peptide is chemically synthesized, anisolated or purified peptide refers to a peptide that does notsubstantially contain a precursor substance or another chemicalsubstance. The phrase “does not substantially contain a cellularmaterial” includes peptide preparations in which the peptide isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a peptide that does notsubstantially contain a cellular material encompasses peptidepreparations that contain less than about 30%, 20%, 10%, 5%, 3%, 2% or1% (dry weight basis) of other cellular materials. When the peptide isrecombinantly produced, an isolated or purified peptide does notsubstantially contain culture medium, which encompasses peptidepreparations that contain culture medium less than about 20%, 10%, or5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptidepreparation. When the peptide is chemically synthesized, an isolated orpurified peptide does not substantially contain a precursor substance orother chemical substances, which encompasses peptide preparations thatcontain a precursor substance or other chemical substances less thanabout 30%, 20%, 10%, 5%, 3%, 2% or 1% (dry weight basis) of the volumeof the peptide preparation. That a particular peptide preparation is anisolated or purified peptide can be confirmed, for example, by theappearance of a single band following sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis and Coomassie Brillliant Bluestaining or such of the gel. In a preferred embodiment, the peptides andpolynucleotides of the present invention are isolated or purified.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein, and refer to polymers of amino acid residues.These terms are applied to also non-naturally occurring amino acidpolymers comprising one or more non-naturally occurring amino acidresidues, in addition to naturally occurring amino acid polymers.Non-naturally occurring amino acids include amino acid analogs, aminoacid mimetics, and such.

The term “amino acid” as used herein refers to naturally occurring aminoacids, as well as amino acid analogs and amino acid mimetics thatfunctions similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose modified after translation in cells (e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine, etc.). The phrase “aminoacid analog” refers to compounds that have the same basic chemicalstructure (an alpha carbon bound to a hydrogen, a carboxy group, anamino group, and an R group) as a naturally occurring amino acid buthave a modified R group or modified backbones (e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium, andsuch). The phrase “amino acid mimetic” refers to chemical compounds thathave different structures but similar functions to general amino acids.Amino acids can be either L-amino acids or D-amino acids, and thepeptides of the present invention are preferably L-amino acid polymers.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein, and refer to a polymer of nucleotides.

The term “composition” used in the present specification is intended toencompass products that include specified ingredients in specifiedamounts, and any products generated directly or indirectly fromcombination of specified ingredients in the specified amounts. When thecomposition is a pharmaceutical composition, the term “composition” isintended to encompass products including active ingredient(s) and inertingredient(s), as well as any products generated directly or indirectlyfrom combination, complexation or aggregation of any two or moreingredients, from dissociation of one or more ingredients, or from othertypes of reactions or interactions of one or more ingredients. Thus, thepharmaceutical compositions of the present invention encompass anycompositions made by admixing compounds or cells of the presentinvention with a pharmaceutically or physiologically acceptable carrier.Without being limited thereto, the terms “pharmaceutically acceptablecarrier” or “physiologically acceptable carrier” used in the presentspecification include liquid or solid bulking agents, diluents,excipients, solvents, and encapsulation materials; and meanpharmaceutically or physiologically acceptable materials, compositions,substances or media.

Unless otherwise specified, the term “cancer” refers to a cancer thatoverexpresses the KOC1 gene; and examples thereof include bladdercancer, cervical cancer, cholangiocellular cancer, chronic myeloidleukemia (CML), colon cancer, rectum cancer, esophagus cancer, diffusegastric cancer, non-small-cell lung cancer, small-cell lung cancer,lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head and neckcancer, soft tissue tumor, testis cancer and such, without being limitedthereto. In an exemplary embodiment, the “cancer” is a cancer thatexpresses KOC1 and HLA-A11, HLA-A33, HLA-A03 and/or HLA-A01.

Unless otherwise specified, the terms “cytotoxic T lymphocyte” and“cytotoxic T cell” and “CTL” are used interchangeably herein. Unlessotherwise specifically indicated, they refer to a sub-group of Tlymphocytes that can recognize non-self cells (for example, tumor/cancercells, virus-infected cells) and induce the death of such cells.

Unless otherwise specified, the term “HLA-A11” refers to the HLA-A11type which includes subtypes such as HLA-A*1101, HLA-A*1102, HLA-A*1103,and HLA-A*1104.

Unless otherwise specified, the term “HLA-A33” refers to the HLA-A33type which includes subtypes such as HLA-A*3303, HLA-A*3301, andHLA-A*3304.

Unless otherwise specified, the term “HLA-A03” refers to the HLA-A03type which includes subtypes such as HLA-A*0301, HLA-A*0302, andHLA-A*0305.

Unless otherwise specified, the term “HLA-A01” refers to the HLA-A01type which includes subtypes such as HLA-A*0101, HLA-A*0102, HLA-A*0103,and HLA-A*0104.

In the context of a subject or patient, the phrase “HLA antigen of asubject (or patient) is HLA-A11” used herein refers to that a subject orpatient has the HLA-A11 antigen gene homozygously or heterozygously asthe MHC (Major Histocompatibility Complex) Class I molecule, and thatthe HLA-A11 antigen is expressed in the cells of the subject or patientas the HLA antigen. Similarly, the phrases “HLA antigen of a subject (orpatient) is HLA-A33”, “HLA antigen of a subject (or patient) is HLA-A03”and “HLA antigen of a subject (or patient) is HLA-A01” used herein referto, respectively, that a subject or patient has the HLA-A33 antigen genehomozygously or heterozygously as the MHC (Major HistocompatibilityComplex) Class I molecule and that the HLA-A33 antigen is expressed asthe HLA antigen in the cells of the subject or patient; that a subjector patient has the HLA-A03 antigen gene homozygously or heterozygouslyas the MHC (Major Histocompatibility Complex) Class I molecule and thatthe HLA-A03 antigen is expressed as the HLA antigen in the cells of thesubject or patient; and that a subject or patient has the HLA-A01antigen gene homozygously or heterozygously as the MHC (MajorHistocompatibility Complex) Class I molecule and that the HLA-A01antigen is expressed as the HLA antigen in the cells of the subject orpatient.

As long as the methods and compositions of the present invention areuseful in the context of cancer “treatment”, the treatment is considered“efficacious” when it achieves clinical advantages, for example,reduction in the size, spreading or metastatic ability of cancer,retardation of cancer progression, alleviation of clinical symptoms ofcancer, prolongation of survival period, suppression of postoperativerecurrence in a subject. When the treatment is applied prophylactically,“efficacious” means that the treatment retards or prevents cancerformation, or prevents or alleviates clinical symptoms of cancer.Effectiveness is determined in relation to any publicly known method fordiagnosing or treating a specific tumor type.

As long as the methods and compositions of the present invention areuseful in the context of cancer “prevention (prophylaxis)”, the term“prevention (prophylaxis)” herein includes any work that eases the loadof cancer-associated mortality or morbidity. Prevention (Prophylaxis)can be carried out at the “primary, secondary and tertiary prevention(prophylaxis) levels”. Whereas the primary prevention (prophylaxis)avoids the development of a disease, prevention (prophylaxis) at thesecondary and tertiary levels encompasses prevention (prophylaxis) ofdisease progression and appearance of symptoms, as well as workingsintended to reduce adverse effects of the existing disease by restoringfunctions and reducing disease-associated complications. Alternately,prevention (prophylaxis) can include alleviation of severity of aspecific disorder, for example, extensive preventive therapy intended toreduce tumor growth and metastasis.

In the context of the present invention, the treatment and/or prevention(prophylaxis) of cancer and/or prevention (prophylaxis) of postoperativerecurrence thereof include either of the events such as inhibition ofcancer cell proliferation, tumor involution or regression, induction ofremission and suppression of cancer development, tumor regression, aswell as reduction or inhibition of metastasis, suppression ofpostoperative recurrence of cancer, and prolongation of survival period.Effective treatment and/or prevention (prophylaxis) of cancer reducemortality, improve prognosis of an individual with cancer, reduce theblood levels of tumor markers, and alleviate detectable symptomsassociated with cancer. For example, alleviation or improvement ofsymptoms constitutes effective treatment and/or prevention(prophylaxis), and includes a condition in which the symptoms are stableor alleviated by 10%, 20%, 30% or more.

In the context of the present invention, the term “antibody” refers toimmunoglobulins and fragments thereof that are specifically reactive toa designated protein or peptide thereof. An antibody can include humanantibodies, primatized antibodies, chimeric antibodies, bispecificantibodies, humanized antibodies, antibodies fused to other proteins orradiolabels, and antibody fragments. Furthermore, an “antibody” hereinis used in the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from two or more intact antibodies, andantibody fragments so long as they exhibit the desired biologicalactivity. An “antibody” may be antibodies of all classes (e.g., IgA,IgD, IgE, IgG and IgM).

Unless otherwise specified, the technical terms and scientific termsused herein all have the same meanings as terms commonly understood byone of ordinary skill in the art to which the present invention belongs.

II. Peptides

HLA-A11 and HLA-A33 are alleles commonly seen in Asians (Sette A, SidneyJ., Immunogenetics 1999, 50: 201-12), and HLA-A03 and HLA-A01 arealleles commonly seen in Caucasians (Cao et al., Hum Immunol 2001;62(9): 1009-30). Thus, an effective method of treating KOC1-expressingcancers for a great population of Asians or Caucasians can be providedby providing KOC1-derived CTL-inducing peptides restricted to HLA-A11,HLA-A33, HLA-A03, or HLA-A01. Thus, the present invention providesKOC1-derived peptides that are capable of inducing CTLs in an HLA-A11-,HLA-A33-, HLA-A03-, or HLA-A01-restrictive manner.

The peptides of the present invention are KOC1-derived peptides that arecapable of inducing CTLs in an HLA-A11-, HLA-A33-, HLA-A03-, orHLA-A01-restrictive manner. Peptides capable of inducing CTLs in anHLA-A11-restrictive manner include peptides having the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32. Similarly,peptides capable of inducing CTLs in an HLA-A33-restrictive mannerinclude peptides having the amino acid sequence selected from among SEQID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85. Similarly,peptides capable of inducing CTLs in an HLA-A03-restrictive mannerinclude peptides having the amino acid sequence selected from among SEQID NOs: 27, 30 and 52. Similarly, peptides capable of inducing CTLs inan HLA-A01-restrictive manner include peptides having the amino acidsequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41.

CTLs having a cytotoxic activity specific to these peptides can beestablished by in vitro stimulation of T cells by dendritic cells (DCs)pulsed with these peptides. The established CTLs show a specificcytotoxic activity against target cells pulsed with each of thepeptides.

The KOC1 gene is overexpressed in cancer cells such as cancer cells in,for example, bladder cancer, cervical cancer, cholangiocellular cancer,chronic myeloid leukemia (CML), colon cancer, rectum cancer, esophaguscancer, diffuse gastric cancer, non-small-cell lung cancer, small-celllung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, headand neck cancer, soft tissue tumor, testis cancer and such, but is notexpressed in most normal organs. It is thus an excellent target forimmunotherapy. Therefore, the peptides of the present invention can besuitably used for cancer immunotherapy. A preferred peptide is anonapeptide (a peptide consisting of 9 amino acid residues) or adecapeptide (a peptide consisting of 10 amino acid residues), and it ismore preferably a peptide consisting of the amino acid sequence selectedfrom among SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52,79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41. For example, a peptidehaving the amino acid sequence of SEQ ID NO: 32 is suitable forinduction of CTLs that show a specific cytotoxic activity against cellsexpressing HLA-A11 and KOC1, and can be suitably used for cancerimmunotherapy for HLA-A11-positive patients. Furthermore, for example, apeptide having the amino acid sequence selected from among SEQ ID NOs:64, 67 and 77 is suitable for induction of CTLs that show a specificcytotoxic activity against cells expressing HLA-A33 and KOC1, and can besuitably used for cancer immunotherapy for HLA-A33-positive patients. Inaddition, for example, a peptide having the amino acid sequence of SEQID NO: 27 is suitable for induction of CTLs that show a specificcytotoxic activity against cells expressing HLA-A03 and KOC1, and can besuitably used for cancer immunotherapy for HLA-A03-positive patients.Additionally, for example, a peptide having the amino acid sequence ofSEQ ID NO: 86 is suitable for induction of CTLs that show a specificcytotoxic activity against cells expressing HLA-A01 and KOC1, and can besuitably used for cancer immunotherapy for HLA-A01-positive patients. Ina more preferred embodiment, the peptide of the present invention is apeptide consisting of the amino acid sequence selected from among SEQ IDNOs: 32, 64, 67, 77, 27, and 86.

For the peptides of the present invention, an additional amino acidresidue(s) can be made to adjoin the amino acid sequence of the peptideof the present invention, as long as the resultant peptides retain theCTL-inducing ability of the original peptide. The additional amino acidresidue(s) may be composed of any types of amino acid(s), as long asthey do not impair the CTL-inducing ability of the original peptide.Therefore, the peptides of the present invention encompass peptideshaving CTL-inducing ability, comprising the amino acid sequence selectedfrom among SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52,79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41. Such peptides are, forexample, less than about 40 amino acids, in many cases less than about20 amino acids, and usually less than about 15 amino acids. Therefore,if the original peptide is a nonapeptide, the peptide of the presentinvention encompasses peptides that are 10 amino-acid long or 11-40amino-acid long, which are produced by adjoining additional aminoacid(s) to the peptide. Furthermore, if the original peptide is adecapeptide, the peptide of the present invention encompasses peptidesthat are 11-40 amino-acid long, which are produced by adjoiningadditional amino acid(s) to the peptide. Such a peptide can be, forexample, a peptide that is 11-20 amino-acid long or a peptide that is11-15 amino-acid long. A preferred example of an additional amino acidresidue(s) is an amino acid residue(s) adjacent to the amino acidsequence of the peptide of the present invention in the full-lengthamino acid sequence of KOC1 (for example, SEQ ID NO: 110). Therefore,the peptides of the present invention encompass peptides comprising theamino acid sequence selected from among SEQ ID NOs: 5, 28, 30, 32, 61,62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and41, and wherein the peptides are peptide fragments of KOC1 and haveCTL-inducing ability.

In general, modifications of one, two or more amino acids in a certainpeptide do not affect the functions of the peptide, or in some caseseven enhance the desired functions of the original peptide. In fact,modified peptides (i.e., peptides composed of the amino acid sequence inwhich one, two or several amino acid residues are modified (i.e.,substituted, deleted, inserted, and/or added) compared to the originalreference sequence) are known to retain the biological activity of theoriginal peptide (Mark et al., Proc Nati Acad Sci USA 1984, 81: 5662-6;Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500;Dalbadie-McFarland et al., Proc Nati Acad Sci USA 1982, 79: 6409-13).Thus, in one embodiment, the peptides of the present invention can bepeptides comprising the amino acid sequence in which one, two or severalamino acids are substituted, deleted, inserted and/or added to the aminoacid sequence selected from among SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63,64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41 andhaving CTL-inducing ability.

On skilled in the art can recognize that individual substitutions to anamino acid sequence that alter a single amino acid or a small percentageof amino acids tend to result in the conservation of the properties ofthe original amino acid side chain(s). Those are frequently referred toas “conservative substitutions” or “conservative modifications”; andmodification of a protein by “conservative substitution” or“conservative modification” may result in a modified protein that hassimilar functions as the original protein. Tables of conservativesubstitutions presenting functionally similar amino acids are well knownin the art. Examples of amino acid side chain characteristics thatfunctionally resemble include, for example, hydrophobic amino acids (A,I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G,H, K, S, T), and side chains having the following functional groups orcharacteristics in common: an aliphatic side-chain (G, A, V, L, I, P); ahydroxyl group containing side-chain (S, T, Y); a sulfur atom containingside-chain (C, M); a carboxylic acid and amide containing side-chain (D,N, E, Q); a base containing side-chain (R, K, H); and an aromaticcontaining side-chain (H, F, Y, W). In addition, the following eightgroups each contain amino acids that are accepted in the art asconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified peptides are also encompassed in peptidesof the present invention. However, peptides of the present invention arenot restricted thereto and can include non-conservative modifications,so long as the modified peptide retains the CTL-inducing ability of theoriginal peptide. Furthermore, modified peptides do not exclude CTLinducible peptides derived from polymorphic variants, interspecieshomologues, and alleles of KOC1.

So long as a peptide retains the CTL-inducing ability of an originalpeptide, one can modify (i.e., substitute, delete, insert and/or add) asmall number (for example, 1, 2 or several) or a small percentage ofamino acids. Herein, the term “several” means 5 or fewer amino acids,for example, 4 or 3 or fewer. The percentage of amino acids to bemodified is preferably 20% or less, more preferably 15% or less, evenmore preferably 10% or less or 1 to 5%.

When used in the context of immunotherapy, peptides of the presentinvention are presented on the surface of a cell or exosome, preferablyas a complex with an HLA antigen. Therefore, it is preferable that thepeptides of the present invention possess high binding affinity to theHLA antigen. To that end, the peptides can be modified by substitution,deletion, insertion, and/or addition of the amino acid residues to yielda modified peptide having improved binding affinity. Since theregularity of the sequences of peptides displayed by binding to HLAantigens is already known (Falk, et al., Immunogenetics 1994 40 232-41;Chujoh, et al., Tissue Antigens 1998: 52: 501-9; Takiguchi, et al.,Tissue Antigens 2000: 55: 296-302.), modifications based on suchregularity can be introduced into the peptides of the present invention.

For example, in peptides having binding affinity for HLA Class I, thesecond amino acid from the N terminus and the C-terminal amino acid aregenerally anchor residues involved in the binding to HLA Class I(Rammensee H G, et al., Immunogenetics. 1995; 41(4): 178-228.). Forexample, for HLA-A11, threonine, valine, isoleucine, leucine,phenylalanine, and tyrosine for the second amino acid from the Nterminus, and lysine and arginine for the C-terminal amino acid areknown as anchor residues with high binding affinity for HLA-A11 (Falk,et al., Immunogenetics 1994, 40: 232-41; Chujoh, et al., Tissue Antigens1998: 52: 501-9). Further, in HLA-A11, there is auxiliary anchorresidues at positions 3 and 7 from the N terminus; and it is known thatleucine, phenylalanine, tyrosine, isoleucine, and alanine are preferredas the third amino acid from the N terminus, and that leucine,isoleucine, tyrosine, valine and phenylalanine are preferred as theseventh amino acid from the N terminus (Falk, et al., Immunogenetics1994, 40: 232-41; Chujoh, et al., Tissue Antigens 1998: 52: 501-9).Thus, to maintain or enhance the HLA-A11-binding affinity, there is apossibility that it is desirable to substitute the second amino acidfrom the N terminus with threonine, valine, isoleucine, leucine,phenylalanine, or tyrosine, and/or to substitute the C-terminal aminoacid with lysine or arginine. Further, there is a possibility that it isalso desirable to substitute the third amino acid from the N terminuswith leucine, phenylalanine, tyrosine, isoleucine, or alanine, and/or tosubstitute the seventh amino acid from the N terminus with leucine,isoleucine, tyrosine, valine or phenylalanine. Thus, peptides withCTL-inducing ability, comprising an amino acid sequence in which, in theamino acid sequence selected from among SEQ ID NOs: 5, 28, 30 and 32,the second amino acid from the N terminus is substituted with threonine,valine, isoleucine, leucine, phenylalanine, or tyrosine; the third aminoacid from the N terminus is substituted with leucine, phenylalanine,tyrosine, isoleucine, or alanine; the seventh amino acid from the Nterminus is substituted with leucine, isoleucine, tyrosine, valine orphenylalanine; and/or the C-terminal amino acid is substituted withlysine or arginine are encompassed by the peptides of the presentinvention. In a preferred embodiment, the peptide of the presentinvention can be a peptide having CTL-inducing ability that consists ofan amino acid sequence in which, in the amino acid sequence selectedfrom among SEQ ID NOs: 5, 28, 30 and 32, the second amino acid from theN terminus is substituted with threonine, valine, isoleucine, leucine,phenylalanine, or tyrosine; the third amino acid from the N terminus issubstituted with leucine, phenylalanine, tyrosine, isoleucine, oralanine; the seventh amino acid from the N terminus is substituted withleucine, isoleucine, tyrosine, valine or phenylalanine; and/or theC-terminal amino acid is substituted with lysine or arginine. That is,the peptides of the present invention encompass peptides havingCTL-inducing ability, which comprise an amino acid sequence in which oneor more substitutions selected from

-   (a) to (d) below are introduced into the amino acid sequence    selected from among SEQ ID NOs: 5, 28, 30 and 32:-   (a) the second amino acid from the N terminus is substituted with    threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine;-   (b) the third amino acid from the N terminus is substituted with    leucine, phenylalanine, tyrosine, isoleucine, or alanine;-   (c) the seventh amino acid from the N terminus is substituted with    leucine, isoleucine, tyrosine, valine or phenylalanine; and-   (d) the C-terminal amino acid is substituted with lysine or    arginine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability that consists of an amino acidsequence in which one or more substitutions selected from (a) to (d)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 5, 28, 30 and 32. In the present invention, the preferrednumber of substitutions is 1, 2, 3 or 4 substitutions selected from (a)to (d) above.

The peptide of the present invention may be a peptide havingCTL-inducing ability, which comprises an amino acid sequence in which,in the amino acid sequence selected from among SEQ ID NOs: 5, 28, 30,and 32, the second amino acid from the N terminus is substituted withthreonine, valine, isoleucine, leucine, phenylalanine, or tyrosine,and/or the C-terminal amino acid is substituted with lysine or arginine.Preferably, the peptide of the present invention may be a peptide havingCTL-inducing ability, which consists of an amino acid sequence in which,in the amino acid sequence selected from among SEQ ID NOs: 5, 28, 30 and32, the second amino acid from the N terminus is substituted withthreonine, valine, isoleucine, leucine, phenylalanine, or tyrosine,and/or the C-terminal amino acid is substituted with lysine or arginine.That is, the peptide of the present invention may be a peptide havingCTL-inducing ability, which comprises an amino acid sequence in whichone or more substitutions selected from (a) and (b) below are introducedinto the amino acid sequence selected from among SEQ ID NOs: 5, 28, 30and 32:

-   -   (a) the second amino acid from the N terminus is substituted        with threonine, valine, isoleucine, leucine, phenylalanine, or        tyrosine; and    -   (b) the C-terminal amino acid is substituted with lysine or        arginine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) to (b)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 5, 28, 30 and 32. In a more preferred embodiment, the secondamino acid from the N terminus is substituted with threonine, valine,isoleucine, or leucine.

In HLA-A33, phenylalanine, tyrosine, alanine, isoleucine, leucine, andvaline for the second amino acid from the N terminus, and lysine andarginine for the C-terminal amino acid are known as anchor residues withhigh binding affinity for HLA-A33 (Falk, et al., Immunogenetics 1994,40: 232-41; Takiguchi, et al., Tissue Antigens 2000, 55: 296-302).Further, in HLA-A33, the first amino acid residue from the N terminus isalso known to function as an anchor residue, and it is known thataspartic acid and glutamic acid is preferred as the first amino acidfrom the N terminus (Falk, et al., Immunogenetics 1994, 40: 232-41;Takiguchi, et al., Tissue Antigens 2000: 55: 296-302). Thus, to maintainor enhance the HLA-A33-binding affinity, there is a possibility that itis desirable to substitute the first amino acid from the N terminus withaspartic acid or glutamic acid, the second amino acid from the Nterminus with phenylalanine, tyrosine, alanine, isoleucine, leucine, orvaline, and/or the C-terminal amino acid with lysine or arginine.Therefore, peptides having CTL-inducing ability, which comprise an aminoacid sequence in which, in the amino acid sequence selected from amongSEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, the firstamino acid from the N terminus is substituted with aspartic acid orglutamic acid, the second amino acid from the N terminus is substitutedwith phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine,and/or the C-terminal amino acid is substituted with lysine or arginineare encompassed by the peptides of the present invention. In a preferredembodiment, the peptide of the present invention may be a peptide havingCTL-inducing ability, which consists of an amino acid sequence in which,in the amino acid sequence selected from among SEQ ID NOs: 61, 62, 63,64, 67, 74, 77, 52, 79, 80, and 85, the first amino acid from the Nterminus is substituted with aspartic acid or glutamic acid, the secondamino acid from the N terminus is substituted with phenylalanine,tyrosine, alanine, isoleucine, leucine, or valine, and/or the C-terminalamino acid is substituted with lysine or arginine. That is, the peptidesof the present invention encompass a peptide having CTL-inducingability, which comprises an amino acid sequence in which one or moresubstitutions selected from (a) to (c) below are introduced into theamino acid sequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67,74, 77, 52, 79, 80, and 85:

-   -   (a) the first amino acid from the N terminus is substituted with        aspartic acid or glutamic acid;    -   (b) the second amino acid from the N terminus is substituted        with phenylalanine, tyrosine, alanine, isoleucine, leucine, or        valine; and    -   (c) the C-terminal amino acid is substituted with arginine or        lysine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) to (c)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85. In thepresent invention, the preferred number of substitutions is 1, 2 or 3substitutions selected from (a) to (c) above.

Furthermore, the peptide of the present invention can be a peptidehaving CTL-inducing ability, which comprises an amino acid sequence inwhich, in the amino acid sequence selected from among SEQ ID NOs: 61,62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, the second amino acid fromthe N terminus is substituted with phenylalanine, tyrosine, alanine,isoleucine, leucine, or valine, and/or the C-terminal amino acid issubstituted with arginine or lysine. Preferably, the peptide of thepresent invention can be a peptide having CTL-inducing ability, whichconsists of an amino acid sequence in which, in the amino acid sequenceselected from among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80,and 85, the second amino acid from the N terminus is substituted withphenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and/orthe C-terminal amino acid is substituted with arginine or lysine. Thatis, the peptide of the present invention can be a peptide havingCTL-inducing ability, which comprises an amino acid sequence in whichone or more substitutions selected from (a) and (b) below are introducedinto the amino acid sequence selected from among SEQ ID NOs: 61, 62, 63,64, 67, 74, 77, 52, 79, 80, and 85:

-   -   (a) the second amino acid from the N terminus is substituted        with phenylalanine, tyrosine, alanine, isoleucine, leucine, or        valine; and    -   (b) the C-terminal amino acid is substituted with arginine or        lysine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) and (b)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85. In a morepreferred embodiment, the second amino acid from the N terminus issubstituted with phenylalanine or tyrosine.

In HLA-A03, leucine, methionine, valine, alanine, isoleucine, serine,and threonine for the second amino acid from the N terminus, andarginine, lysine, tyrosine, and phenylalanine for the C-terminal aminoacid are known as anchor residues with high binding affinity for HLA-A03(Kubo R T. et al., J Immunol. 1994 Apr. 15; 152(8): 3913-24; Sidney Jet. al., Hum Immunol. 1996 February; 45(2): 79-93; Gambacorti-PasseriniC et al., Clin Cancer Res. 1997 May; 3(5): 675-83). Thus, to maintain orenhance the HLA-A03-binding affinity, there is a possibility that it ispreferable to substitute the second amino acid from the N terminus withleucine, methionine, valine, alanine, isoleucine, serine or threonine,and/or the C-terminal amino acid with arginine, lysine, tyrosine orphenylalanine. Therefore, peptides having CTL-inducing ability, whichcomprise an amino acid sequence in which, in the amino acid sequenceselected from among SEQ ID NOs: 27, 30, and 52, the second amino acidfrom the N terminus is substituted with leucine, methionine, valine,alanine, isoleucine, serine or threonine, and/or the C-terminal aminoacid is substituted with arginine, lysine, tyrosine or phenylalanine areencompassed by the peptides of the present invention. In a preferredembodiment, the peptides of the present invention may be a peptidehaving CTL-inducing ability, which consists of an amino acid sequence inwhich, in the amino acid sequence selected from among SEQ ID NOs: 27,30, and 52, the second amino acid from the N terminus is substitutedwith leucine, methionine, valine, alanine, isoleucine, serine orthreonine, and/or the C-terminal amino acid is substituted witharginine, lysine, tyrosine or phenylalanine. That is, the peptides ofthe present invention encompass a peptide having CTL-inducing ability,which comprises an amino acid sequence in which one or moresubstitutions selected from (a) and (b) below are introduced into theamino acid sequence selected from among SEQ ID NOs: 27, 30, and 52:

-   -   (a) the second amino acid from the N terminus is substituted        with leucine, methionine, valine, alanine, isoleucine, serine or        threonine; and    -   (b) the C-terminal amino acid is substituted with arginine,        lysine, tyrosine or phenylalanine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) and (b)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 27, 30, and 52. In a more preferred embodiment, the secondamino acid from the N terminus is substituted with leucine, methionineor valine. In the present invention, the preferred number ofsubstitutions is 1 or 2 substitutions selected from (a) and (b) above.

In HLA-A01, aspartic acid and glutamic acid for the third amino acidfrom the N terminus, and tyrosine for the C-terminal amino acid areknown as anchor residues with high binding affinity for HLA-A01.Further, it is known that there are auxiliary anchor residues atposition 2 from the N terminus for HLA-A01 and that threonine and serineare preferred as the second amino acid from the N terminus (Kubo, R. TJournal of Immunology 1994, 152: 3913; Gambacorti-Passerini, C. ClinicalCancer Research 1997, 3: 675-83; Falk, K. Immunogenetics 1994, 40:238-41). Thus, to maintain or enhance the HLA-A01-binding affinity,there is a possibility that it is desirable to substitute the thirdamino acid from the N terminus with aspartic acid or glutamic acid,and/or the C-terminal amino acid with tyrosine. Another possibility isthat it is desirable to substitute the second amino acid from the Nterminus with threonine or serine. Therefore, peptides havingCTL-inducing ability, which comprise an amino acid sequence in which, inthe amino acid sequence selected from among SEQ ID NOs: 86, 87, 90, 92,46, 95 and 41, the second amino acid from the N terminus is substitutedwith threonine or serine, the third amino acid from the N terminus issubstituted with aspartic acid or glutamic acid, and/or the C-terminalamino acid is substituted with tyrosine are encompassed by the peptidesof the present invention. In a preferred embodiment, the peptide of thepresent invention may be a peptide having CTL-inducing ability, whichconsists of an amino acid sequence in which, in the amino acid sequenceselected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41, thesecond amino acid from the N terminus is substituted with threonine orserine, the third amino acid from the N terminus is substituted withaspartic acid or glutamic acid, and/or the C-terminal amino acid issubstituted with tyrosine. That is, the peptides of the presentinvention encompass a peptide having CTL-inducing ability, whichcomprises an amino acid sequence in which one or more substitutionsselected from (a) to (c) below are introduced into the amino acidsequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41:

-   -   (a) the second amino acid from the N terminus is substituted        with threonine or serine;    -   (b) the third amino acid from the N terminus is substituted with        aspartic acid or glutamic acid; and    -   (c) the C-terminal amino acid is substituted with tyrosine.

In a preferred embodiment, the peptide of the present invention can be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) to (c)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41. In the present invention, thepreferred number of substitutions is 1, 2 or 3 substitutions selectedfrom (a) to (c) above.

Furthermore, the peptide of the present invention may be a peptidehaving CTL-inducing ability, which comprises an amino acid sequence inwhich, in the amino acid sequence selected from among SEQ ID NOs: 86,87, 90, 92, 46, 95 and 41, the third amino acid from the N terminus issubstituted with aspartic acid or glutamic acid, and/or the C-terminalamino acid is substituted with tyrosine. Preferably, the peptide of thepresent invention may be a peptide having CTL-inducing ability, whichconsists of an amino acid sequence in which, in the amino acid sequenceselected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41, the thirdamino acid from the N terminus is substituted with aspartic acid orglutamic acid, and/or the C-terminal amino acid is substituted withtyrosine. That is, the peptide of the present invention can be a peptidehaving CTL-inducing ability, which comprises an amino acid sequence inwhich one or more substitutions selected from (a) and (b) below areintroduced into the amino acid sequence selected from among SEQ ID NOs:86, 87, 90, 92, 46, 95 and 41:

-   -   (a) the third amino acid from the N terminus is substituted with        aspartic acid or glutamic acid; and    -   (b) the C-terminal amino acid is substituted with tyrosine.

In a preferred embodiment, the peptide of the present invention may be apeptide having CTL-inducing ability, which consists of an amino acidsequence in which one or more substitutions selected from (a) to (b)above are introduced into the amino acid sequence selected from amongSEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

Substitution(s) may be introduced into amino acid(s) not only at theanchor site(s), but also at a position(s) of potential T cell receptor(TCR) recognition site(s) of the peptides. Several research studies havedemonstrated that a peptide that has amino acid substitutions, such asCAP1, p₍₂₆₄₋₂₇₂₎, Her-2/neu₍₃₆₉₋₃₇₇₎ or gp100₍₂₀₉₋₂₁₇₎, may have equalto or better activity than that of the original peptide (Zaremba et al.Cancer Res. 57, 4570-7, 1997; T. K. Hoffmann et al. J Immunol. (2002)February 1, 168(3): 1338-47; S. O. Dionne et al. Cancer Immunolimmunother. (2003) 52: 199-206; and S. O. Dionne et al. CancerImmunology, Immunotherapy (2004) 53, 307-14).

The present invention also contemplates that one, two or several aminoacids can be added to the N terminus and/or C terminus of the peptidesof the present invention (for example, peptides consisting of the aminoacid sequence selected from among SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63,64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41). Suchmodified peptides that retain CTL-inducing ability are also included inthe present invention. For example, when a peptide in which one, two orseveral amino acids are added to the N terminus and/or C terminus of apeptide consisting of the amino acid sequence selected from among SEQ IDNOs: 32, 64, 67, 77, 27 and 86 is contacted with an APC(s), it isincorporated into the APC(s) and processed to become a peptideconsisting of the amino acid sequence selected from among SEQ ID NOs:32, 64, 67, 77, 27 and 86. It can then induce CTLs through presentationon the cell surface of an APC via the antigen presentation pathway. Morespecifically, peptides of the present invention can be peptides in whichone, two or several amino acids are added to either or both of the Nterminus and C terminus.

However, when the amino acid sequence of a peptide is identical to aportion of the amino acid sequence of an endogenous or exogenous proteinhaving a different function, side effects such as autoimmune disordersand/or allergic symptoms against specific substances may be induced.Therefore, it is preferable to perform homology searches using availabledatabases to avoid situations in which the amino acid sequence of thepeptide matches the amino acid sequence of another protein. When itbecomes clear from the homology searches that no peptide exists with asfew as 1 or 2 amino acid differences as compared to the objectivepeptide, the objective peptide can be modified in order to increase itsbinding affinity with HLA antigens, and/or increase its CTL-inducingability without danger of such side effects.

Peptides in which one, two or several amino acids of a peptide of thepresent invention are modified are predicted to be able to retainCTL-inducing ability of the original peptide; however, it is preferableto verify the CTL-inducing ability of the modified peptides. Herein, the“peptide having CTL-inducing ability (CTL inducibility)” refers to apeptide that induces CTLs through APCs stimulated with the peptide. “CTLinduction” includes induction of differentiation into CTLs, induction ofCTL activation, induction of CTL proliferation, induction of CTL'scytotoxic activity, induction of CTL-mediated dissolution of targetcells, and induction of increase of IFN-gamma production of CTLs.

The CTL-inducing ability can be confirmed by inducing and stimulatingAPCs that retain an HLA antigen (for example, B lymphocytes,macrophages, and dendritic cells) with a peptide, and mixing it withCD8-positive T cells; and then measuring IFN-gamma released by CTLsagainst the target cells. For the APCs, human peripheral bloodmononuclear leukocyte-derived dendritic cells can be preferably used. Asa reaction system, transgenic animals generated to express an HLAantigen can be used. Alternatively, for example, the target cells may beradio-labelled with ⁵¹Cr or such, and the cytotoxic activity of thepeptide-induced CTLs may be calculated from the radioactivity emittedfrom the target cells. Alternatively, in the presence ofpeptide-stimulated APCs, it is possible to evaluate the CTL-inducingability by measuring the IFN-gamma produced and released by CTLs, andvisualizing the inhibition zone on the media using anti-IFN-gammamonoclonal antibodies.

In addition to the modifications above, the peptides of the presentinvention can be linked to other peptides as long as the resultantlinked peptide retains the CTL-inducing ability. An example of anappropriate peptide to be linked with the peptides of the presentinvention includes a TAA-derived CTL-inducing peptide. Further, thepeptides of the present invention can also be linked with each other.Suitable linkers for use in linking peptides are known in the art, andfor example, linkers such as AAY (P. M. Daftarian et al., J Trans Med2007, 5:26), AAA, NKRK (SEQ ID NO: 111) (R. P. M. Sutmuller et al., JImmunol. 2000, 165: 7308-15), or K (S. Ota et at, Can Res. 62, 1471-6,K. S. Kawamura et al., J Immunol. 2002, 168: 5709-15) can be used.Peptides can be linked in various arrangements (for example, catenulate,repeated, etc.), and one can also link three or more peptides.

The peptides of the present invention can also be linked to othersubstances as long as the resultant linked peptide retains theCTL-inducing ability. Examples of an appropriate substance to be linkedwith a peptide of the present invention include, for example, a peptide,a lipid, a sugar or sugar chain, an acetyl group, and anaturally-occurring or synthetic polymer. The peptides of the presentinvention can be modified by glycosylation, side-chain oxidation,phosphorylation or such, as long as their CTL-inducing ability is notimpaired. One can also perform such types of modifications to conferadditional functions (for example, targeting function and deliveryfunction) or to stabilize the peptide.

For example, to increase the in vivo stability of a peptide, it is knownin the art to introduce D-amino acids, amino acid mimetics ornon-naturally occurring amino acids, and this concept may also beapplied to peptides of the present invention. Peptide stability can beassayed by several methods. For example, stability can be tested byusing a peptidase as well as various biological media such as humanplasma and serum (see, e.g., Verhoef et al., Eur J Drug MetabPharmacokin 1986, 11: 291-302).

Further, as stated above, among the modified peptides in which one, two,or several amino acid residues have been substituted, deleted, insertedand/or added, those having the same or higher activity as compared tooriginal peptides can be screened for or selected. Thus, the presentinvention also provides methods of screening for or selecting modifiedpeptides that have the same or higher activity than that of the originalpeptide. Specifically, the present invention provides a method ofscreening for a peptide having CTL-inducing ability, wherein the methodcomprises the steps of:

-   (a) generating candidate sequences consisting of an amino acid    sequence in which one, two, or several amino acid residues are    substituted, deleted, inserted and/or added to the original amino    acid sequence consisting of the amino acid sequence selected from    among SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79,    80, 85, 27, 86, 87, 90, 92, 46, 95 and 41;-   (b) selecting from among the candidate sequences generated in (a), a    candidate sequence that does not have a significant homology    (sequence identity) with any known human gene product other than    KOC1;-   (c) contacting a peptide consisting of the candidate sequence    selected in (b) with APCs;-   (d) contacting the APCs of (c) with CD8-positive T cells; and-   (e) selecting a peptide that has an equal to or higher CTL-inducing    ability than that of a peptide consisting of the original amino acid    sequence.

Herein, the peptide of the present invention is also described as a“KOC1 peptide(s)” or a “KOC1 polypeptide(s)”.

III. Preparation of Peptides of the Present Invention

Well known techniques can be used to prepare peptides of the presentinvention. For example, recombinant DNA technology or chemical synthesiscan be used to prepare peptides of the present invention. Peptides ofthe present invention can be synthesized individually, or as longerpolypeptides including two or more peptides. Peptides of the presentinvention can be isolated from host cells or synthesis reaction productsafter they are produced in the host cells using recombinant DNAtechnology or after they are chemically synthesized. That is, peptidesof the present invention can be purified or isolated so as not tosubstantially contain other host-cell proteins and fragments thereof, orany other chemical substances.

The peptides of the present invention may contain modifications, such asglycosylation, side chain oxidation, or phosphorylation provided suchmodifications do not destroy the biological activity of the originalpeptide. Other illustrative modifications include incorporation ofD-amino acids or other amino acid mimetics that may be used, forexample, to increase the serum half life of the peptides.

A peptide of the present invention can be obtained through chemicalsynthesis based on the selected amino acid sequence. Examples ofconventional peptide synthesis methods that can be adapted to thesynthesis include the methods described in the documents below:

(i) Peptide Synthesis, Interscience, New York, 1966;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) “Peptide Synthesis” (in Japanese), Maruzen Co., 1975;

(iv) “Basics and Experiment of Peptide Synthesis” (in Japanese), MaruzenCo., 1985;

(v) “Development of Pharmaceuticals” (in Japanese), Continued Vol. 14(peptide synthesis), Hirokawa, 1991;

(vi) WO99/67288; and

(vii) Barany G. & Merrifield R. B., Peptides Vol. 2, Solid Phase PeptideSynthesis, Academic Press, New York, 1980, 100-118.

Alternatively, the peptides of the present invention can be obtainedadapting any known genetic engineering methods for producing peptides(e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss &Curtiss, Methods in Enzymology (Wu et al.) 1983, 101: 347-62). Forexample, first, a suitable vector harboring a polynucleotide encodingthe peptide of the present invention in an expressible form (e.g.,downstream of a regulatory sequence corresponding to a promotersequence) is prepared and transformed into a suitable host cell. Thehost cell is then cultured to produce the peptide of the presentinvention. The peptide of the present invention can also be produced invitro using an in vitro translation system.

IV. Polynucleotides

The present invention also provides a polynucleotide which encodes anyof the peptides of the present invention. These include polynucleotidesderived from the naturally occurring KOC1 gene (e.g., GenBank AccessionNo. NM_006547 (SEQ ID NO: 109)) as well as those having a conservativelymodified nucleotide sequence thereof. Herein, the phrase “conservativelymodified nucleotide sequence” refers to sequences which encode identicalor essentially identical amino acid sequences. Due to the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG, andGCU all encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described above without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations”, whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a peptide also describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a peptide is implicitly described in each disclosedsequence.

The polynucleotide of the present invention can be composed of DNA, RNA,and derivatives thereof. A DNA is suitably composed of bases such as A,T, C, and G, and T is replaced by U in an RNA.

The polynucleotide of the present invention can encode multiple peptidesof the present invention with or without intervening amino acidsequences in between. For example, the intervening amino acid sequencecan provide a cleavage site (e.g., enzyme recognition sequence) of thepolynucleotide or the translated peptides. Furthermore, thepolynucleotide can include any additional sequences to the codingsequence encoding the peptide of the present invention. For example, thepolynucleotide can be a recombinant polynucleotide that includesregulatory sequences required for the expression of the peptide or canbe an expression vector (e.g., plasmid) with marker genes and such. Ingeneral, such recombinant polynucleotides can be prepared by themanipulation of polynucleotides through conventional recombinanttechniques using, for example, polymerases and endonucleases.

Both recombinant and chemical synthesis techniques can be used toproduce the polynucleotides of the present invention. For example, apolynucleotide can be produced by insertion into an appropriate vector,which can be expressed when transfected into a competent cell.Alternatively, a polynucleotide can be amplified using PCR techniques orexpression in suitable hosts (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1989). Alternatively, a polynucleotide can be synthesized using thesolid phase techniques, as described in Beaucage S L & Iyer R P,Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

V. Exosomes

The present invention further provides intracellular vesicles, referredto as exosomes, that present complexes formed between the peptides ofthe present invention and HLA antigens on their surface. Exosomes can beprepared, for example, using the methods detailed in JPH11-510507 andWO99/03499, and can be prepared using APCs obtained from patients whoare subject to treatment and/or prevention (prophylaxis). The exosomesof the present invention can be inoculated as vaccines, in a fashionsimilar to the peptides of the present invention.

The type of the HLA antigens included in the above-described complexesmust match that of the subject in need of treatment and/or prevention(prophylaxis). For example, in Asian populations, HLA-A11 (for example,HLA-A*1101) and HLA-A33 (for example, HLA-A*3303) are alleles widely andgenerally seen in Asian populations, and these HLA antigen types areconsidered to be suitable for treatment in Asian patients. Further,HLA-A03 (for example, HLA-A*0301) and HLA-A01 (for example, HLA-A*0101)are alleles widely and generally seen in Caucasian populations, andthese HLA antigen types are considered to be suitable for treatment inCaucasian patients. Typically in clinical practice, it is possible toselect an appropriate peptide that has a high level of binding affinityfor a specific HLA antigen or that has CTL-inducing ability by antigenpresentation mediated by a specific HLA antigen, by studying in advancethe HLA antigen type of the patient in need of treatment.

The exosomes of the present invention present on their surface a complexof a peptide of the present invention and HLA-A11, HLA-A33, HLA-A03 orHLA-A01. When the HLA that forms a complex with a peptide of the presentinvention is HLA-A11, the peptide of the present invention is preferablya peptide having the amino acid sequence selected from among SEQ ID NOs:5, 28, 30 and 32 or a modified peptide thereof, and more preferably apeptide consisting of the amino acid sequence selected from among SEQ IDNOs: 5, 28, 30 and 32 or a modified peptide thereof. Further, when theHLA that forms a complex with a peptide of the present invention isHLA-A33, the peptide of the present invention is preferably a peptidehaving the amino acid sequence selected from among SEQ ID NOs: 61, 62,63, 64, 67, 74, 77, 52, 79, 80 and 85 or a modified peptide thereof, andmore preferably a peptide consisting of the amino acid sequence selectedfrom among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85 ora modified peptide thereof. Further, when the HLA that forms a complexwith a peptide of the present invention is HLA-A03, the peptide of thepresent invention is preferably a peptide having the amino acid sequenceselected from among SEQ ID NOs: 27, 30 and 52 or a modified peptidethereof, and more preferably a peptide consisting of the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 or a modifiedpeptide thereof. Further, when the HLA that forms a complex with apeptide of the present invention is HLA-A01, the peptide of the presentinvention is preferably a peptide having the amino acid sequenceselected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41 or amodified peptide thereof, and more preferably a peptide consisting ofthe amino acid sequence selected from among SEQ ID NOs: 86, 87, 90, 92,46, 95 and 41 or a modified peptide thereof.

VI. Antigen-Presenting Cells (APCs)

The present invention further provides APCs that present on theirsurface complexes formed between HLA antigens and peptides of thepresent invention. Alternatively, the present invention provides APCshaving on their cell surface complexes formed between HLA antigens andpeptides of the present invention. The APCs of the present invention canbe isolated APCs. When used in the context of cells (APCs, CTLs, etc.),the term “isolated” means that the cells are separated from another typeof cells. The APCs of the present invention may be APCs induced fromAPCs derived from the patient to be subjected to treatment and/orprevention (prophylaxis), and can be administered as a vaccine bythemselves or in combination with other drugs including a peptide(s), anexosome(s) or a CTL(s) of the present invention.

The APCs of the present invention are not limited to specific types ofcells, and include cells known to present proteinaceous antigens ontheir cell surface so as to be recognized by lymphocytes, for example,dendritic cells (DCs), Langerhans cells, macrophages, B cells, andactivated T cells. Since DC is a representative APC that has thestrongest CTL-inducing activity among APCs, DCs can be preferably usedas the APCs of the present invention. In the present invention, thepreferable DC is an isolated DC derived from human. Further, it is notnecessary for the APCs of the present invention to be homogeneous, andthey can be mixtures of multiple types of cells having anantigen-presenting function and can be mixtures of APCs each of whichpresents different types of the peptides of the present invention.

For example, APCs of the present invention can be obtained by isolatingDCs from peripheral blood mononuclear cells and then stimulating them invitro with the peptides of the present invention. When the peptide ofthe present invention is administered to a subject, APCs presenting thepeptide of the present invention are induced in the body of the subject.Therefore, after the peptides of the present invention are administeredto a subject, the APCs of the present invention can be obtained bycollecting APCs from the subject. Alternatively, the APCs of the presentinvention can be obtained by contacting APCs collected from a subjectwith a peptide of the present invention.

In order to induce an immune response against KOC1-expressing cancercells in a subject, the APCs of the present invention can beadministered to the subject by themselves or in combination with otherdrugs including peptide(s), exosome(s) or CTL(s) of the presentinvention. For example, the ex vivo administration can comprise thefollowing steps of:

(a) collecting APCs from a first subject;

(b) contacting the APCs of step (a) with a peptide; and

(c) administering the APCs of step (b) to a second subject.

The first subject and the second subject may be the same individual, ormay be different individuals. When the first subject and the secondsubject are different individuals, it is preferable that the HLAs of thefirst subject and the second subject are of the same type. The APCobtained in step (b) above can be a vaccine for cancer treatment and/orprevention (prophylaxis).

The APCs of the present invention obtained by a method such as describedabove have CTL-inducing ability. The term “CTL-inducing ability (CTLinducibility)” used in the context of an APC(s) refers to the ability ofthe APC to be able to induce a CTL(s) when placed in contact with aCD8-positive T cell(s). Further, the “CTL-inducing ability (CTLinducibility)” includes the ability of an APC to induce CTL activation,the ability of an APC to induce CTL proliferation, the ability of an APCto facilitate CTL-mediated dissolution of target cells, and the abilityof an APC to increase CTL-mediated IFN-gamma production. The CTL(s)induced by the APC of the present invention is a CTL(s) specific to KOC1and demonstrates a specific cytotoxic activity against KOC1-expressingcells.

In addition to the above-described methods, the APCs of the presentinvention can be prepared by introducing a polynucleotide encoding apeptide of the present invention into APCs in vitro. The polynucleotideto be introduced can be in the form of DNA or RNA. The method ofintroduction is not particularly limited, and examples thereof includevarious methods conventionally performed in the art such as lipofection,electroporation and the calcium phosphate method. More specifically,methods described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161:5607-13; J Exp Med 1996, 184: 465-72, and JP2000-509281 can be used. Byintroducing a polynucleotide encoding a peptide of the present inventioninto an APC, the polynucleotide is transcribed and translated in thecell, and then the produced peptide is processed by MHC Class I andproceeds through a presentation pathway to present the peptide of thepresent invention on the cell surface of the APC.

In a preferred embodiment, the APC of the present invention presents onits cell surface a complex formed between a peptide of the presentinvention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (morepreferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or HLA-A01(more preferably HLA-A*0101). When the HLA that forms a complex with apeptide of the present invention is HLA-A11, the peptide of the presentinvention is preferably a peptide having the amino acid sequenceselected from among SEQ ID NOs: 5, 28, 30 and 32 or a modified peptidethereof, and more preferably a peptide consisting of the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32. When the HLAthat forms a complex with a peptide of the present invention is HLA-A33,the peptide of the present invention is preferably a peptide having theamino acid sequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67,74, 77, 52, 79, 80 and 85 or a modified peptide thereof, and morepreferably a peptide consisting of the amino acid sequence selected fromamong SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85. Whenthe HLA that forms a complex with a peptide of the present invention isHLA-A03, the peptide of the present invention is preferably a peptidehaving the amino acid sequence selected from among SEQ ID NOs: 27, 30and 52 or a modified peptide thereof, and more preferably a peptideconsisting of the amino acid sequence selected from among SEQ ID NOs:27, 30 and 52. When the HLA that forms a complex with a peptide of thepresent invention is HLA-A01, the peptide of the present invention ispreferably a peptide having the amino acid sequence selected from amongSEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41 or a modified peptide thereof,and more preferably a peptide consisting of the amino acid sequenceselected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

The APC(s) of the present invention is preferably an APC(s) induced by amethod comprising a step described (a) or (b) below:

-   (a) contacting an APC(s) expressing at least one HLA selected from    among HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably    HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) and HLA-A01 (more    preferably HLA-A*0101) with a peptide of the present invention; or-   (b) introducing a polynucleotide encoding a peptide of the present    invention into an APC(s) expressing at least one HLA selected from    among HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably    HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) and HLA-A01 (more    preferably HLA-A*0101).

The peptide of the present invention to be contacted with theHLA-A11-expressing APC(s) is preferably a peptide having the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32 or a modifiedpeptide thereof, and more preferably a peptide consisting of the aminoacid sequence selected from among SEQ ID NOs: 5, 28, 30 and 32.

The peptide of the present invention to be contacted with theHLA-A33-expressing APC(s) is preferably a peptide having the aminosequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52,79, 80 and 85 or a modified peptide thereof, and more preferably apeptide consisting of the amino sequence selected from among SEQ ID NOs:61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85.

The peptide of the present invention to be contacted with theHLA-A03-expressing APC(s) is preferably a peptide having the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 or a modifiedpeptide thereof, and more preferably a peptide consisting of the aminoacid sequence selected from among SEQ ID NOs: 27, 30 and 52.

The peptide of the present invention to be contacted with theHLA-A01-expressing APC(s) is preferably a peptide having the amino acidsequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41or a modified peptide thereof, and more preferably a peptide consistingof the amino acid sequence selected from among SEQ ID NOs: 86, 87, 90,92, 46, 95 and 41.

The present invention provides use of a peptide of the present inventionfor the manufacture of a pharmaceutical composition that induces anAPC(s) having CTL-inducing ability. In addition, the present inventionprovides a method or process of manufacturing a pharmaceuticalcomposition that induces an APC(s) having CTL-inducing ability. Further,the present invention provides a peptide of the present invention forinducing an APC(s) having CTL-inducing ability.

VII. Cytotoxic T Lymphocytes (CTLs)

The CTL induced by a peptide of the present invention can be used as avaccine in a similar manner to the peptide of the present invention forenhancing an immune response targeting KOC1-expressing cell in vivo.Thus, the present invention provides CTLs that are induced or activatedby a peptide of the present invention. The CTLs of the present inventionare CTLs that target a peptide of the present invention, and are capableof binding to a complex of a peptide of the present invention and an HLAantigen. Binding of a CTL to the complex is mediated via a T cellreceptor (TCR) present on the cell surface of the CTL. The CTLs of thepresent invention can be isolated CTLs. The preferable CTLs are isolatedCTLs of human origin. The CTLs of the present invention do not have tobe homogeneous, and can be mixtures of CTLs each of which targetsdifferent types of peptides of the present invention.

The CTLs of the present invention can be obtained by (1) administering apeptide of the present invention to a subject, (2) stimulating APCs andCD8-positive T cells, or peripheral blood mononuclear cells (PBMCs)derived from a subject with a peptide of the present invention in vitro,(3) contacting in vitro CD8-positive T cells or PBMCs with APCs orexosomes that present on their surface a complex of an HLA antigen and apeptide of the present invention, or (4) introducing into CD8-positive Tcells a vector comprising a polynucleotide encoding each subunit of a Tcell receptor (TCR) capable of binding to a peptide of the presentinvention presented on cell surface via an HLA antigen. The exosomes andAPCs used in the method of (2) or (3) above can be prepared by methodsdescribed in the “V. Exosomes” and “VI. Antigen-presenting cells (APCs)”sections, respectively, and the details of the method of (4) above willbe described in the “VIII. T cell receptor (TCR)” section.

The CTLs of the present invention can be administered by themselves topatients who are subject to treatment and/or prevention (prophylaxis),or in combination with other drugs including peptide(s), APC(s) orexosome(s) of the present invention for the purpose of regulatingeffects. Further, the CTLs of the present invention can be CTLs inducedfrom CD8-positive T cells derived from the patients who are subject toadministration of the CTLs. The CTLs of the present invention actspecifically on target cells that present the peptides of the presentinvention, for example, the same peptides used to induce the CTLs of thepresent invention. The target cells may be cells that endogenouslyexpress KOC1, such as cancer cells, or cells transfected with the KOC1gene. Cells that present a peptide of the present invention on theircell surface due to stimulation by the peptide can become a target ofattack by the CTLs of the present invention. The cells targeted by theCTLs of the present invention are preferably cells that are positive forat least one of HLA-A11 (more preferably HLA-A*1101), HLA-A33 (morepreferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301), andHLA-A01 (more preferably HLA-A*0101).

In a preferred embodiment, the CTLs of the present invention targetspecifically cells that express both KOC1 and at least one HLA selectedfrom among HLA-A11 (more preferably HLA-A*1101), HLA-A33 (morepreferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301), andHLA-A01 (more preferably HLA-A*0101). In the present invention, thecells targeted by the CTLs can be cells that have any of the alleles ofHLA-A11, HLA-A33, HLA-A03, and HLA-A01 homozygously or heterozygously.

Herein, that the CTL “targets” cells refers to CTL recognition of cellsthat present on their cell surface a complex of HLA and a peptide of thepresent invention and demonstration of a cytotoxic activity against thecells. Further, “specifically target” refers to that the CTLsdemonstrate a cytotoxic activity against those cells, but do not show adamaging activity to other cells. The expression “recognize cells” usedin the context of CTLs refers to binding to a complex of HLA and apeptide of the present invention presented on cell surface via its TCR,and demonstrating a specific cytotoxic activity against the cell.Therefore, the CTLs of the present invention are preferably CTLs thatcan bind via TCR to a complex formed between a peptide of the presentinvention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (morepreferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or HLA-A01(more preferably HLA-A*0101) presented on cell surface.

Furthermore, the CTLs of the present invention are preferably CTLsinduced by a method comprising a step described in (a) or (b) below:

-   (a) contacting in vitro CD8-positive T cells with APCs or exosomes    that present on their surface a complex of a peptide of the present    invention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more    preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or    HLA-A01 (more preferably HLA-A*0101); or-   (b) introducing into CD8-positive T cells a polynucleotide encoding    each subunit of a TCR capable of binding to a peptide of the present    invention presented on cell surface by HLA-A11 (more preferably    HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more    preferably HLA-A*0301) or HLA-A01 (more preferably HLA-A*0101).

VIII. T Cell Receptors (TCRs)

The present invention also provides compositions comprising apolynucleotide encoding each subunit of a TCR capable of binding to apeptide of the present invention presented on cell surface by an HLAantigen, and methods of using the same. The polynucleotide confersCD8-positive T cells with specificity against KOC1-expressing cancercells through expression of a TCR capable of binding to a peptide of thepresent invention presented on cell surface by an HLA antigen.Polynucleotides encoding an alpha chain(s) and a beta chain(s) can beidentified as the TCR subunit of the CTL induced by a peptide of thepresent invention by using known methods in the art (WO2007/032255 andMorgan et al., J Immunol, 171, 3288 (2003)). For example, PCR methodsare preferred for TCR analysis. Without being limited thereto, PCRprimers for analysis may be, for example, a primer set(s) foramplification by combining the 5′ side primer and the 3′ side primer(s)below:

5′ side primer: 5′-R Primer (SEQ ID NO: 105)(5′-gtctaccaggcattcgcttcat-3′) 3′ side primers:TCR-alpha-chain C-region-specific 3-TRa-C Primer (SEQ ID NO: 106)(5′-tcagctggaccacagccgcagcgt-3′) TCR-beta-chain C1-region-specific3-TRb-C1 Primer (SEQ ID NO: 107) (5′-tcagaaatcctttctcttgac-3′) orTCR-beta-chain C2-region-specific 3-TR-beta-C2 Primer (SEQ ID NO: 108)(5′-ctagcctctggaatcctttctctt-3′)

The TCRs formed by introducing the identified polynucleotides intoCD8-positive T cells can bind with high binding affinity to the targetcells that present a peptide of the present invention, and mediatesefficient killing of the target cells presenting a peptide of thepresent invention in vivo and in vitro.

A polynucleotide encoding each TCR subunit can be incorporated into anappropriate vector, for example, retrovirus vector. These vectors arewell known in the art. The polynucleotide or a vector comprising thereofin an expressible form can be introduced into a CD8-positive T cell, forexample, a CD8-positive T cell derived from a patient. The presentinvention provides off-the-shelf compositions that allow rapid and easyproduction of modified T cells that have superior cancer cell-killingproperties by rapid modification of the patient's own T cells (or Tcells derived from another subject).

Herein, a specific TCR is a TCR that can confer a specific cytotoxicactivity against target cells by specifically recognizing a complex of apeptide of the present invention and an HLA antigen presented on thesurface of the target cell when the TCR is present on the surface of aCD8-positive T cell. Specific recognition of the above-described complexcan be confirmed by any known method, and preferable examples thereofinclude HLA multimer staining analysis using HLA molecules and peptidesof the present invention and ELISPOT assay methods. SpecificTCR-mediated recognition of target cell by T cell introduced with theabove-described polynucleotide and signal transduction in the cell canbe confirmed by carrying out an ELISPOT assay. When the above-describedTCR is present on the surface of a CD8-positive T cell, whether the TCRcan confer a target cell-specific cytotoxic activity against theCD8-positive T cell can also be confirmed by known methods. Preferablemethods include, for example, measuring the cytotoxic activity againsttarget cells by a chrome release assay method or such. The presentinvention provides, in the context of HLA-A11, CTLs prepared bytransforming CD8-positive T cells with a polynucleotide encoding eachsubunit of TCR that binds to, for example, a peptide having the aminoacid sequence selected from among SEQ ID NOs: 5, 28, 30 and 32.

The present invention provides, in the context of HLA-A33, CTLs preparedby transforming CD8-positive T cells with a polynucleotide encoding eachsubunit of TCR that binds to, for example, a peptide having the aminoacid sequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67, 74,77, 52, 79, 80 and 85.

The present invention provides, in the context of HLA-A03, CTLs preparedby transforming CD8-positive T cells with a polynucleotide encoding eachsubunit of TCR that binds to, for example, a peptide having the aminoacid sequence selected from among SEQ ID NOs: 27, 30 and 52.

The present invention provides, in the context of HLA-A01, CTLs preparedby transforming CD8-positive T cells with a polynucleotide encoding eachsubunit of TCR that binds to, for example, a peptide having the aminoacid sequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and41.

The transformed CTLs are capable of homing in vivo and may be propagatedby a well-known in vitro culturing method (for example, Kawakami et al.,J Immunol., 142, 3452-61 (1989)). The CTLs of the present invention canbe used to form an immunogenic composition useful for disease treatmentor prevention (prophylaxis) in a patient in need of treatment orprevention (prophylaxis) (the contents are incorporated herein forreference WO2006/031221).

IX. Pharmaceutical Compositions

The present invention further provides compositions or pharmaceuticalcompositions, comprising at least one active ingredient selected frombelow:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC of the present invention;

(d) an exosome of the present invention; and

(e) a CTL of the present invention.

The pharmaceutical compositions of the present invention can comprise asneeded a carrier(s), an excipient(s) or such commonly used inpharmaceuticals without particular limitations, in addition to theactive ingredient(s) described above. An example of a carrier that canbe used in a pharmaceutical composition of the present inventionincludes sterilized water, physiological saline, phosphate buffer,culture fluid and such. Therefore, the present invention also providespharmaceutical compositions, comprising at least one active ingredientselected from (a) to (e) below and a pharmaceutically acceptablecarrier:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC of the present invention;

(d) an exosome of the present invention; and

(e) a CTL of the present invention.

Further, the pharmaceutical compositions of the present invention cancomprise, as needed, stabilizers, suspensions, preservatives,surfactants, solubilizing agents, pH adjusters, aggregation inhibitorsand such.

The KOC1 expression significantly up-regulates in cancer cells comparedwith normal tissues. Thus, a peptide of the present invention or apolynucleotide encoding the peptide can be used to treat and/or preventcancer, and/or prevent postoperative recurrence thereof. Therefore, thepresent invention provides pharmaceutical compositions for treatingand/or preventing cancer, and/or preventing postoperative recurrencethereof, comprising one or more types of peptides or polynucleotides ofthe present invention as an active ingredient. Alternatively, thepeptides of the present invention can be made to be presented on thesurface of exosomes or APCs for use as pharmaceutical compositions. Inaddition, CTLs of the present invention targeting any one of thepeptides of the present invention can also be used as an activeingredient of the pharmaceutical compositions of the present invention.The pharmaceutical compositions of the present invention may comprise atherapeutically effective amount or a pharmaceutically effective amountof the above-described active ingredient.

The pharmaceutical compositions of the present invention may also beused as a vaccine. In the context of the present invention, the term“vaccine” (also called “immunogenic composition”) refers to acomposition that has a function of inducing an immune response thatleads to antitumor action when inoculated into an animal. Thus, apharmaceutical composition of the present invention can be used toinduce an immune response that leads to antitumor action. The immuneresponse induced by a peptide, a polynucleotide, an APC, a CTL and apharmaceutical composition of the present invention is not particularlylimited as long as it is an immune response that leads to antitumoraction, and examples include induction of cancer cell-specific CTLs andinduction of cancer cell-specific cytotoxic activity.

The pharmaceutical compositions of the present invention can be used totreat and/or prevent cancer, and/or prevent postoperative recurrencethereof in human subjects or patients. The pharmaceutical compositionsof the present invention can be used preferably to a subject positivefor at least one HLA selected from among HLA-A11, HLA-A33, HLA-A03 andHLA-A01. Further, the pharmaceutical compositions of the presentinvention can be used preferably to treat and/or prevent cancersexpressing KOC1 and at least one HLA selected from among HLA-A11,HLA-A33, HLA-A03 and HLA-A01, and/or prevent postoperative recurrencethereof.

In another embodiment, the present invention provides use of an activeingredient selected from below in the manufacture of a pharmaceuticalcomposition for treating or preventing cancer:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides an activeingredient selected from below for use in treating or preventing cancer:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides a method orprocess for manufacturing a pharmaceutical composition for treating orpreventing cancer, wherein the method or process comprises a step offormulating at least one active ingredient selected from below with apharmaceutically or physiologically acceptable carrier:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

In another embodiment, the present invention further provides a methodor process for manufacturing a pharmaceutical composition for treatingor preventing cancer, wherein the method or process comprises a step ofmixing an active ingredient selected from below with a pharmaceuticallyor physiologically acceptable carrier:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

In another embodiment, the present invention further provides a methodfor treating or preventing cancer, which comprises a step ofadministering to a subject at least one active ingredient selected frombelow:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

In the present invention, peptides having the amino acid sequenceselected from among SEQ ID NOs: 5, 28, 30 and 32 are identified asHLA-A11-restricted epitope peptides that can induce a potent andspecific immune response. Therefore, pharmaceutical compositions of thepresent invention comprising at least one peptide having the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32 are suitableparticularly for administration to a subject having HLA-A11 (forexample, HLA-A*1101) as an HLA antigen. The same applies topharmaceutical compositions comprising a polynucleotide encoding any ofthese peptides (i.e., polynucleotides of the present invention), an APCor exosome that presents these peptides (i.e., APCs or exosomes of thepresent invention), or a CTL targeting these peptides (i.e., CTLs of thepresent invention). That is, pharmaceutical compositions comprising anactive ingredient in association with a peptide having the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32 are suitablefor administration to subjects having HLA-A11 (i.e., HLA-A11-positivesubjects). In a more preferred embodiment, the pharmaceuticalcomposition of the present invention is a pharmaceutical compositionthat comprises a peptide having the amino acid sequence of SEQ ID NO:32.

Similarly, in the present invention, peptides having the amino acidsequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52,79, 80 and 85 are identified as HLA-A33-restricted epitope peptides thatcan induce a potent and specific immune response. Therefore,pharmaceutical compositions of the present invention comprising at leastone peptide having the amino acid sequence selected from among SEQ IDNOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85 are suitableparticularly for administration to a subject having HLA-A33 (forexample, HLA-A*3303) as an HLA antigen. The same applies topharmaceutical compositions comprising a polynucleotide encoding any ofthese peptides (i.e., polynucleotides of the present invention), an APCor exosome that presents these peptides (i.e., APCs or exosomes of thepresent invention), or a CTL targeting these peptides (i.e., CTLs of thepresent invention). That is, pharmaceutical compositions comprising anactive ingredient in association with a peptide having the amino acidsequence selected from among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52,79, 80 and 85 are suitable for administration to subjects having HLA-A33(i.e., HLA-A33-positive subjects). In a more preferred embodiment, thepharmaceutical composition of the present invention is a pharmaceuticalcomposition that comprises at least one peptide having the amino acidsequence selected from among SEQ ID NOs: 64, 67, and 77.

Similarly, in the present invention, peptides having the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 are identified asHLA-A03-restricted epitope peptides that can induce a potent andspecific immune response. Therefore, pharmaceutical compositions of thepresent invention comprising at least one peptide having the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 are suitableparticularly for administration to a subject having HLA-A03 (forexample, HLA-A*0301) as an HLA antigen. The same applies topharmaceutical compositions comprising a polynucleotide encoding any ofthese peptides (i.e., polynucleotides of the present invention), an APCor exosome that presents these peptides (i.e., APCs or exosomes of thepresent invention), or a CTL targeting these peptides (i.e., CTLs of thepresent invention). That is, pharmaceutical compositions comprising anactive ingredient in association with a peptide having the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 are suitable foradministration to subjects having HLA-A03 (i.e., HLA-A03-positivesubjects). In a more preferred embodiment, the pharmaceuticalcomposition of the present invention is a pharmaceutical compositionthat comprises a peptide having the amino acid sequence of SEQ ID NO:27.

Similarly, in the present invention, peptides having the amino acidsequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41are identified as HLA-A01-restricted epitope peptides that can induce apotent and specific immune response. Therefore, pharmaceuticalcompositions of the present invention comprising at least one peptidehaving the amino acid sequence selected from among SEQ ID NOs: 86, 87,90, 92, 46, 95 and 41 are suitable particularly for administration to asubject having HLA-A01 (for example, HLA-A*0101) as an HLA antigen. Thesame applies to pharmaceutical compositions comprising a polynucleotideencoding any of these peptides (i.e., polynucleotides of the presentinvention), an APC or exosome that presents these peptides (i.e., APCsor exosomes of the present invention), or a CTL targeting these peptides(i.e., CTLs of the present invention). That is, pharmaceuticalcompositions comprising an active ingredient in association with apeptide having the amino acid sequence selected from among SEQ ID NOs:86, 87, 90, 92, 46, 95 and 41 are suitable for administration tosubjects having HLA-A01 (i.e., HLA-A01-positive subjects). In a morepreferred embodiment, the pharmaceutical composition of the presentinvention is a pharmaceutical composition that comprises a peptidehaving the amino acid sequence of SEQ ID NO: 86.

Cancers to be treated and/or prevented by pharmaceutical compositions ofthe present invention are not particularly limited as long as they arecancers that express KOC1, and include bladder cancer, cervical cancer,cholangiocellular cancer, chronic myeloid leukemia (CML), colon cancer,rectum cancer, esophagus cancer, diffuse gastric cancer, non-small-celllung cancer, small-cell lung cancer, lymphoma, osteosarcoma, ovariancancer, kidney cancer, head and neck cancer, soft tissue tumor, testiscancer and such. It is preferable to use the pharmaceutical compositionof the present invention in subjects that homozygously or heterozygouslyhave an HLA allele selected from among HLA-A11, HLA-A33, HLA-A03 andHLA-A01.

In addition to the active ingredients described above, thepharmaceutical compositions of the present invention can comprise theother peptides that have the ability to induce CTLs against cancer cells(for example, the other TAA-derived CTL-inducing peptides), the otherpolynucleotides encoding the other peptides, the other cells thatpresent the other peptides, or such.

The pharmaceutical compositions of the present invention may alsooptionally comprise the other therapeutic substances as an activeingredient, as long as they do not inhibit the anti-tumor effects of theabove-described active ingredients such as peptides of the presentinvention. For example, the pharmaceutical compositions of the presentinvention may optionally comprise anti-inflammatory compositions,analgesics, chemotherapeutics and the like. In addition to including theother therapeutic substances to a pharmaceutical composition of thepresent invention itself, one can also administer the pharmaceuticalcomposition of the present invention sequentially or concurrently withone or more other pharmaceutical compositions. The dose of thepharmaceutical composition of the present invention and the otherpharmaceutical compositions depend on, for example, the type ofpharmaceutical composition used and the disease being treated, as wellas the scheduling and routes of administration.

It should be understood that in consideration of the formulation type,the pharmaceutical composition of the present invention may includeother components conventional in the art, in addition to the ingredientsspecifically mentioned herein.

The present invention also provides articles of manufacture or kits thatcomprise a pharmaceutical composition of the present invention. Thearticles of manufacture or kits of the present invention can include acontainer that houses the pharmaceutical composition of the presentinvention. An example of an appropriate container includes a bottle, avial or a test tube, but is not limited thereto. The container may beformed of various materials such as glass or plastic. A label may beattached to the container, and the disease or disease state to which thepharmaceutical composition of the present invention should be used maybe described in the label. The label may also indicate directions foradministration and such.

The articles of manufacture or kits of the present invention may furthercomprise a second container that houses pharmaceutically acceptablediluents optionally, in addition to the container that houses thepharmaceutical composition of the present invention. The articles ofmanufacture or kits of the present invention may further comprise theother materials desirable from a commercial standpoint and the user'sperspective, such as the other buffers, diluents, filters, injectionneedles, syringes, package inserts with instructions for use.

As needed, the pharmaceutical composition of the present invention canbe provided in a pack or dispenser device that can contain one or moreunits of dosage forms containing active ingredients. The pack caninclude, for example, a metallic foil or a plastic foil such as ablister pack. Instructions for administration can be attached to thepack or dispenser device.

(1) Pharmaceutical Compositions Comprising Peptide(s) as an ActiveIngredient

The pharmaceutical composition comprising a peptide of the presentinvention can be formulated by conventional formulation methods asneeded. The pharmaceutical compositions of the present invention maycomprise as needed in addition to the peptide of the present invention,carriers, excipients and such commonly used in pharmaceuticals withoutparticular limitations. Examples of carriers that can be used inpharmaceutical compositions of the present invention include sterilizedwater (for example, water for injection), physiological saline,phosphate buffer, phosphate buffered saline, Tris buffered saline, 0.3%glycine, culture fluid, and such. Further, the pharmaceuticalcompositions of the present invention may comprise as neededstabilizers, suspensions, preservatives, surfactants, solubilizingagents, pH adjusters, aggregation inhibitors, and such. Thepharmaceutical compositions of the present invention can induce specificimmunity against KOC1-expressing cancer cells, and thus can be appliedfor the purpose of cancer treatment or prevention (prophylaxis).

For example, the pharmaceutical compositions of the present inventioncan be prepared by dissolving in pharmaceutically or physiologicallyacceptable water-soluble carriers such as sterilized water (for example,water for injection), physiological saline, phosphate buffer, phosphatebuffered saline, and Tris buffered saline and adding, as needed,stabilizers, suspensions, preservatives, surfactants, solubilizingagents, pH adjusters, aggregation inhibitors and such, and thensterilizing the peptide solution. The method of sterilizing a peptidesolution is not particularly limited, and is preferably carried out byfiltration sterilization. Filtration sterilization can be performedusing, for example, a filtration sterilization filter of 0.22 micro-m orless in pore diameter. The filtration-sterilized peptide solution can beadministered to a subject, for example, as an injection, without beinglimited thereto. The pharmaceutical compositions of the presentinvention may be prepared as a freeze-dried formulation by freeze-dryingthe above-described peptide solution. The freeze-dried formulation canbe prepared by filling the peptide solution prepared as described aboveinto an appropriate container such as an ampule, a vial or a plasticcontainer, followed by freeze drying and encapsulation into thecontainer with a wash-sterilized rubber plug or such after pressurerecovery. The freeze-dried formulation can be administered to a subjectafter it is re-dissolved in pharmaceutically or physiologicallyacceptable water-soluble carriers such as sterilized water (for example,water for injection), physiological saline, phosphate buffer, phosphatebuffered saline, Tris buffered saline and such before administration.Preferred examples of pharmaceutical compositions of the presentinvention include injections of such a filtration-sterilized peptidesolution, and freeze-dried formulations that result from freeze-dryingthe peptide solution. The present invention further encompasses kitscomprising such a freeze-dried formulation and re-dissolving solution.The present invention also encompasses kits comprising a container thathouses the freeze-dried formulation, which is a pharmaceuticalcomposition of the present invention, and a container that houses are-dissolving solution thereof.

The pharmaceutical compositions of the present invention can comprise acombination of two or more types of the peptides of the presentinvention. The combination of peptides can take a cocktail form of mixedpeptides, or can be conjugated with each other using standardtechniques. For example, peptides can be chemically linked or expressedas single fusion polypeptide sequences. By administering a peptide ofthe present invention, the peptide is presented on APCs by an HLAantigen at a high density, and then subsequently CTLs that reactspecifically to a complex formed between the presented peptide and theHLA antigen are induced. Alternatively, APCs (for example, DCs) areremoved from a subject, and subsequently stimulated with peptides of thepresent invention to obtain APCs that present any of the peptides of thepresent invention on their cell surface. These APCs are re-administeredto a subject to induce CTLs in the subject, and as a result, theaggressiveness towards KOC1-expressing cancer cells can be increased.

The pharmaceutical compositions of the present invention may alsocomprise an adjuvant known for effectively establishing cellularimmunity. An adjuvant refers to a compound that enhances the immuneresponse against an antigen that has immunological activity whenadministered together (or successively) with the antigen. Knownadjuvants described in literatures, for example, Clin Microbiol Rev1994, 7: 277-89, can be used. Examples of a suitable adjuvant includealuminum salts (aluminum phosphate, aluminum hydroxide, aluminumoxyhydroxide and such), alum, cholera toxin, Salmonella toxin,Incomplete Freund's adjuvant (IFA), Complete Freund's adjuvant (CFA),ISCOMatrix, GM-CSF and other immunostimulatory cytokines,oligodeoxynucleotide containing the CpG motif (CpG7909 and such),oil-in-water emulsions, Saponin or its derivatives (QS21 and such),lipopolysaccharide such as Lipid A or its derivatives (MPL, RC529, GLA,E6020 and such), lipopeptides, lactoferrin, flagellin, double-strandedRNA or its derivatives (poli IC and such), bacterial DNA,imidazoquinolines (Imiquimod, R848 and such), C-type lectin ligand(trehalose-6,6′-dibehenate (TDB) and such), CD1d ligand(alpha-galactosylceramide and such), squalene emulsions (MF59, AS03,AF03 and such), PLGA, and such, without being limited thereto. Theadjuvant may be contained in another container separate from thepharmaceutical composition comprising a peptide of the present inventionin the kits comprising the pharmaceutical composition of the presentinvention. In this case, the adjuvant and the pharmaceutical compositionmay be administered to a subject in succession, or mixed togetherimmediately before administration to a subject. Such kits comprising apharmaceutical composition comprising a peptide of the present inventionand an adjuvant are also provided by the present invention. When thepharmaceutical composition of the present invention is a freeze-driedformulation, the kit can further comprise a re-dissolving solution.Further, the present invention provides kits comprising a container thathouses a pharmaceutical composition of the present invention and acontainer that stores an adjuvant. The kit can further comprise asneeded a container that stores the re-dissolving solution.

When an oil adjuvant is used as an adjuvant, the pharmaceuticalcomposition of the present invention may be prepared as an emulsion.Emulsions can be prepared, for example, by mixing and stirring thepeptide solution prepared as described above and an oil adjuvant. Thepeptide solution may be one that has been re-dissolved afterfreeze-drying. The emulsion may be either of the W/O-type emulsion andO/W-type emulsion, and the W/O-type emulsion is preferred for obtaininga high immune response-enhancing effect. IFA can be preferably used asan oil adjuvant, without being limited thereto. Preparation of anemulsion can be carried out immediately before administration to asubject, and in this case, the pharmaceutical composition of the presentinvention may be provided as a kit comprising the peptide solution ofthe present invention and an oil adjuvant. When the pharmaceuticalcomposition of the present invention is a freeze-dried formulation, thekit can further comprise a re-dissolving solution.

Further, the pharmaceutical composition of the present invention may bea liposome formulation within which a peptide of the present inventionis encapsulated, a granular formulation in which a peptide is bound tobeads with several micrometers in diameter, or a formulation in which alipid is bound to a peptide.

In another embodiment of the present invention, the peptide of thepresent invention may also be administered in the form of apharmaceutically acceptable salt. Preferred examples of salts includesalts with alkali metals (lithium, potassium, sodium and such), saltswith alkaline-earth metals (calcium, magnesium and such), salts withother metals (copper, iron, zinc, manganese and such), salts withorganic bases, salts with amines, salts with organic acids (acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid and such), and salts with inorganic acids(hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid,nitric acid and such). The phrase “pharmaceutically acceptable salt”used herein refers to a salt that retains the biological, physiological,pharmacological and pharmaceutical efficacy and property of thecompound. Therefore, pharmaceutical compositions comprising apharmaceutically acceptable salt of a peptide of the present inventionare also encompassed by the present invention. Further, the “peptide ofthe present invention” also encompasses, in addition to the freepeptide, pharmaceutically acceptable salts thereof.

In some embodiments, the pharmaceutical compositions of the presentinvention may further include a component which primes CTLs. Lipids havebeen identified as substances capable of priming CTLs in vivo againstviral antigens. For example, palmitic acid residues can be attached tothe epsilon- and alpha-amino groups of a lysine residue and then linkedto a peptide of the present invention. The lipidated peptide can then beadministered either directly in a micelle or particle, incorporated intoa liposome, or emulsified in an adjuvant. As another example of lipidpriming of CTL responses, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used toprime CTLs when covalently attached to an appropriate peptide (see,e.g., Deres et al., Nature 1989, 342: 561-4).

Examples of suitable methods for administering the peptides orpharmaceutical compositions of the present invention include oral,epidermal, subcutaneous, intramuscular, intraosseous, peritoneal, andintravenous injections, as well as systemic administration or localadministration to the vicinity of the targeted sites, but are notlimited thereto. A preferred administration method includes subcutaneousinjection to the vicinity of lymph nodes such as the armpit or groin.The administration can be performed by single administration or boostedby multiple administrations. The peptides of the present invention canbe administered to a subject in a therapeutically or pharmaceuticallyeffective amount for treating cancer or in a therapeutically orpharmaceutically effective amount for inducing immunity (morespecifically CTLs) against KOC1-expressing cancer cells. The dose of thepeptides of the present invention can be appropriately adjustedaccording to the disease to be treated, the patient's age and weight,the method of administration and such. For each of the peptides of thepresent invention, the dose is usually 0.001 mg-1000 mg, for example,0.01 mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1 mg-10 mg,for example, 0.5 mg-5 mg. The dosing interval can be once every severaldays to several months, and for example, the dosing can be done in aonce-per-week interval. A skilled artisan can appropriately select asuitable dose (dosage).

In a preferred embodiment, the pharmaceutical compositions of thepresent invention comprise a therapeutically effective amount of apeptide of the present invention and a pharmaceutically orphysiologically acceptable carrier. In another embodiment, thepharmaceutical compositions of the present invention comprise atherapeutically effective amount of a peptide of the present invention,a pharmaceutically or physiologically acceptable carrier, and anadjuvant. The pharmaceutical compositions of the present invention cancomprise 0.001 mg-1000 mg, preferably 0.01 mg-100 mg, more preferably0.1 mg-30 mg, even more preferably 0.1 mg-10 mg, for example, 0.5 mg-5mg of a peptide of the present invention. When a pharmaceuticalcomposition of the present invention is an injection, it can comprise apeptide of the present invention at a concentration of 0.001 mg/ml-1000mg/ml, preferably 0.01 mg/ml-100 mg/ml, more preferably 0.1 mg/ml-30mg/ml, even more preferably 0.1 mg/ml-10 mg/ml, for example, 0.5 mg/ml-5mg/ml. In this case, for example, 0.1 to 5 ml, preferably 0.5 ml to 2 mlof the pharmaceutical composition of the present invention can beadministered to a subject by injection.

Further, the present invention provides methods of treating and/orpreventing cancer and/or preventing postoperative recurrence thereof,which comprise administering to a subject a therapeutically effectiveamount of a peptide of the present invention or a pharmaceuticalcomposition of the present invention. As described above, the peptidesof the present invention can be administered to a subject in a singledose of usually 0.001 mg-1000 mg, for example, 0.01 mg-100 mg, forexample, 0.1 mg-30 mg, for example, 0.1 mg-10 mg, or for example, 0.5mg-5 mg. In a preferred embodiment, the peptides of the presentinvention are administered to a subject together with an adjuvant.Further, the dosing interval can be once every several days to severalmonths, preferably once every several days to every month, for example,once every week or once every two weeks.

(2) Pharmaceutical Compositions Containing Polynucleotides as the ActiveIngredient

The pharmaceutical compositions of the present invention can alsocontain polynucleotides encoding the peptides of the present inventionin an expressible form. Herein, the phrase “in an expressible form”means that the polynucleotide, when introduced into a cell, will beexpressed as a peptide of the present invention. In an exemplifiedembodiment, the sequence of the polynucleotide of the present inventionincludes regulatory elements necessary for expression of the peptide ofthe present invention. The polynucleotide(s) of the present inventioncan be equipped with a sequence necessary to achieve stable insertioninto the genome of the target cell (see, e.g., Thomas K R & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombinationcassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8;U.S. Pat. Nos. 5,580,859, 5,589,466, 5,804,566, 5,739,118, 5,736,524,5,679,647; and WO98/04720. Examples of DNA-based delivery technologiesinclude “naked DNA”, facilitated (bupivacaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

The peptides of the present invention can also be expressed by viral orbacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. For example, as a vector toexpress the peptide of the present invention, vaccinia virus can beused. Upon introduction into a host, the recombinant vaccinia virusexpresses the immunogenic peptide, and thereby elicits an immuneresponse. Vaccinia vectors and methods useful in immunization protocolsare described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 1991, 351: 456-60. A wide variety of other vectors useful fortherapeutic administration or immunization, e.g., adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71;Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo2000, 14: 571-85.

Delivery of a polynucleotide of the present invention into a patient canbe either direct, in which case the patient can be directly exposed to avector harboring the polynucleotide of the present invention, orindirect, in which case, cells are first transformed with the vectorharboring the polynucleotide of the present invention in vitro, then thecells are transplanted into the patient. These two approaches are known,respectively, as in vivo and ex vivo gene therapies.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan,Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62:191-217; Trends in Biotechnology 1993, 11(5): 155-215. Methods commonlyknown in the art of recombinant DNA technology which can also be usedfor the present invention are described in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, N Y, 1993; andKrieger, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, N Y, 1990.

Similar to peptide administration, administration of polynucleotides maybe performed by oral, intradermal, subcutaneous, intravenous,intramuscular, intraosseous and/or peritoneal injection, and such.Polynucleotide administration can be a systemic administration or alocal administration to the vicinity of the targeted sites. Theadministration can be performed by single administration or boosted bymultiple administrations. The polynucleotides of the present inventioncan be administered to a subject in a therapeutically orpharmaceutically effective dose for inducing immunity (more specificallyCTLs) against KOC1-expressing cancer cells, or in a therapeutically orpharmaceutically effective dose for treating cancer. The dose of apolynucleotide in a suitable carrier or the dose of a polynucleotide incells transformed with a polynucleotide encoding a peptide of thepresent invention can be appropriately adjusted according to the diseaseto be treated, the patient's age and weight, the method ofadministration and such, and this may be usually 0.001 mg-1000 mg, forexample, 0.01 mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1mg-10 mg, or for example, 0.5 mg-5 mg. The dosing interval can be onceevery several days to several months, and for example, the dosing can bedone in a once-per-week interval. A skilled artisan can appropriatelyselect a suitable dose (dosage).

X. Methods of Using Peptides, Exosomes, APCs and CTLs

The peptides and polynucleotides of the present invention can be used toinduce APCs and CTLs. CTLs can also be induced using the exosomes andAPCs of the present invention. The peptides, polynucleotides, exosomes,and APCs can be used in combination with any other compound(s) as longas their CTL-inducing ability is not inhibited. Therefore, CTLs of thepresent invention can be induced using a pharmaceutical compositioncomprising any of the peptides, polynucleotides, APCs and exosomes ofthe present invention. Further, APCs of the present invention can beinduced using a pharmaceutical composition comprising a peptide orpolynucleotide of the present invention.

(1) Methods of Inducing APCs

The present invention provides methods of inducing APCs havingCTL-inducing ability, using a peptide(s) or polynucleotide(s) of thepresent invention.

The methods of the present invention comprise a step of contacting anAPC with a peptide of the present invention in vitro, ex vivo, or invivo. For example, a method of contacting APCs with the peptide ex vivomay comprise the steps below:

(a) collecting APCs from a subject; and

(b) contacting the APCs of step (a) with a peptide of the presentinvention.

The above-described APCs are not limited to a particular type of cell,and cells known to present a proteinaceous antigen on their cell surfaceto be recognized by lymphocytes, for example, DCs, Langerhans cells,macrophages, B cells, and activated T cells can be used. DCs have themost potent CTL-inducing ability among APCs, and thus it is preferableto use DCs. Any peptides of the present invention can be used bythemselves or in combination with other peptides of the presentinvention. Further, peptides of the present invention can be used incombination with other CTL-inducing peptides (for example, otherTAA-derived CTL-inducing peptides).

Meanwhile, when a peptide of the present invention is administered to asubject, APCs are contacted with the peptide in vivo, and as a result,APCs having a high CTL-inducing ability are induced in the body of thesubject. Therefore, the methods of the present invention may comprise astep of administering a peptide of the present invention to a subject.Similarly, when a polynucleotide of the present invention isadministered to a subject in an expressible form, a peptide of thepresent invention is expressed in vivo, the expressed peptide iscontacted with APCs in vivo, and as a result APCs having a highCTL-inducing ability are induced in the body of the subject. Therefore,the present invention may also comprise a step of administering apolynucleotide of the present invention to a subject.

In order to induce APCs having CTL-inducing ability, the presentinvention may comprise a step of introducing a polynucleotide of thepresent invention into APCs. For example, the method may comprise thesteps below:

-   -   (a) collecting APCs from a subject; and    -   (b) introducing a polynucleotide encoding a peptide of the        present invention into the APCs of step (a).        Step (b) can be performed as described in the above “VI.        Antigen-presenting cells (APCs)” section.

Thus, in one embodiment, the present invention provides a method ofinducing APCs having CTL-inducing ability, which comprises the step (a)or (b) below:

(a) contacting APCs with a peptide of the present invention; and

(b) introducing a polynucleotide encoding a peptide of the presentinvention into APCs.

Furthermore, the present invention provides a method of preparing APCshaving CTL-inducing ability, which comprises the step (a) or (b) below:

(a) contacting APCs with a peptide of the present invention; or

(b) introducing a polynucleotide encoding a peptide of the presentinvention into APCs.

The above-described methods can be performed in vitro, ex vivo, or invivo, and it is preferable to perform them in vitro or ex vivo. APCsused in the above-described methods may be derived from a subjectscheduled for administration of the induced APCs, or they may be derivedfrom a different subject. When APCs derived from a subject (donor)different from the subject scheduled for administration are used, thesubject of administration and the donor must have the identical HLAtype. In the methods of the present invention, when a peptide having theamino acid sequence selected from among SEQ ID NOs: 5, 28, 30 and 32 ora modified peptide thereof is used as a peptide of the presentinvention, the HLA type is preferably HLA-A11 (more preferablyHLA-A*1101) in both the subject of administration and the donor.Alternatively, APCs used in the above-described methods are preferablyAPCs that express HLA-A11 (more preferably HLA-A*1101). Similarly, whena peptide having the amino acid sequence selected from among SEQ ID NOs:61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85 or a modified peptidethereof is used as a peptide of the present invention, the HLA ispreferably HLA-A33 (more preferably HLA-A*3303) in both the subject ofadministration and the donor. Alternatively, APCs used in theabove-described methods are preferably APCs that express HLA-A33 (morepreferably HLA-A*3303). Similarly, when a peptide having the amino acidsequence selected from among SEQ ID NOs: 27, 30 and 52 or a modifiedpeptide thereof is used as a peptide of the present invention, the HLAtype is preferably HLA-A03 (more preferably HLA-A*0301) in both thesubject of administration and the donor. Alternatively, APCs used in theabove-described methods are preferably APCs that express HLA-A03 (morepreferably HLA-A*0301). Similarly, when a peptide comprising the aminoacid sequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and41 or a modified peptide thereof is used as a peptide of the presentinvention, the HLA type is preferably HLA-A01 (more preferablyHLA-A*0101) in both the subject of administration and the donor.Alternatively, APCs used in the above-described methods are preferablyAPCs that express HLA-A01 (more preferably HLA-A*0101). The APCs can beprepared using known methods from PBMCs after PBMCs are separated fromblood collected from a donor by a specific gravity centrifugal method orsuch.

In another embodiment, the present invention also providespharmaceutical compositions that comprise a peptide of the presentinvention or a polynucleotide encoding the peptide for inducing anAPC(s) having CTL-inducing ability.

Alternatively, the present invention further provides use of a peptideof the present invention or a polynucleotide encoding the peptide in themanufacture of a pharmaceutical composition for inducing an APC(s)having CTL-inducing ability.

Alternatively, the present invention further provides peptides of thepresent invention or polynucleotides encoding the peptides for use inthe induction of an APC(s) having CTL-inducing ability.

Alternatively, the present invention further provides methods orprocesses of manufacturing a pharmaceutical composition for inducing anAPC(s), wherein the method or process comprises a step of formulating apeptide of the present invention or a polynucleotide encoding thepeptide with a pharmaceutically or physiologically acceptable carrier.

In another embodiment, the present invention further provides methods orprocesses of manufacturing a pharmaceutical composition for inducing anAPC(s) having CTL-inducing ability, wherein the method or processcomprises a step of mixing a peptide of the present invention or apolynucleotide encoding the peptide with a pharmaceutically orphysiologically acceptable carrier.

APCs induced by the methods of the present invention can induce CTLsspecific to KOC1 (i.e., CTLs of the present invention).

(2) Methods of Inducing CTLs

The present invention also provides methods of inducing CTLs usingpeptides, polynucleotides, exosomes or APCs of the present invention.The present invention further provides methods of inducing CTLs usingone or more polynucleotides encoding a polypeptide(s) that can form a Tcell receptor (TCR) (i.e., TCR subunit) capable of recognizing a complexof a peptide of present invention and an HLA antigen. Preferably, themethods of inducing CTLs comprise at least one steps selected frombelow:

-   -   (a) contacting CD8-positive T cells with antigen-presenting        cells that present on their surface a complex of an HLA antigen        and a peptide of present invention;    -   (b) contacting CD8-positive T cells with exosomes that present        on its surface a complex of an HLA antigen and a peptide of        present invention; and    -   (c) introducing into CD8-positive T cells one or more        polynucleotides encoding a polypeptide(s) that can form a TCR        capable of recognizing a complex of a peptide of present        invention and an HLA antigen.

When a peptide(s), a polynucleotide(s), an exosome(s) or an APC(s) ofthe present invention is administered to a subject, CTLs are induced inthe body of the subject and the strength of the immune responsetargeting KOC1-expressing cancer cells is enhanced. Therefore, themethods of the present invention may comprise a step of administering apeptide(s), a polynucleotide(s), an APC(s) or an exosome(s) of thepresent invention to a subject.

Alternatively, CTLs can be induced by using them in vitro or ex vivo.For example, the methods of the present invention may include thefollowing steps:

(a) collecting APCs from a subject;

(b) contacting the APCs of step (a) with a peptide of the presentinvention; and

(c) co-culturing the APCs of step (b) with CD8-positive T cells.

The induced CTLs may be returned to the subject afterwards.

The APCs to be co-cultured with the CD8-positive T cells in step (c)above can also be prepared by introducing into APCs a polynucleotideencoding a peptide of the present invention as described above in the“VI. Antigen-presenting cells (APCs)” section. However, the APCs to beused in the methods of the present invention are not limited thereto,and any APCs that present on their surface a complex of an HLA antigenand a peptide of the present invention can be used.

In the methods of the present invention, instead of such APCs, exosomesthat present on their surface a complex of an HLA antigen and a peptideof the present invention can also be used. That is, the methods of thepresent invention can comprise a step of co-culturing with exosomes thatpresent on their surface a complex of an HLA antigen and a peptide ofthe present invention. Such exosomes can be prepared by theabove-described methods in the “V. Exosomes” section.

Further, CTLs can also be induced by introducing into a CD8-positive Tcell a vector comprising a polynucleotide encoding each subunit of a TCRcapable of binding to a peptide of the present invention presented by anHLA antigen on the cell surface. Such transformation can be carried outas described above in the “VIII. T cell receptors (TCRs)” section.

Accordingly, in one embodiment, the present invention provides methodsof inducing CTLs, comprising a step selected from below:

-   -   (a) co-culturing CD8-positive T cells with APCs that present on        their surface a complex of an HLA antigen and a peptide of        present invention;    -   (b) co-culturing CD8-positive T cells with exosomes that present        on their surface a complex of an HLA antigen and a peptide of        present invention; and    -   (c) introducing into CD8-positive T cells, a vector comprising a        polynucleotide encoding each subunit of a TCR capable of binding        to a peptide of the present invention presented by an HLA        antigen on a cell surface.

The above-described methods can be performed in vitro, ex vivo, or invivo, and it is preferable to perform them in vitro or ex vivo. APCs orexosomes and CD8-positive T cells used in the above-described methodsmay be derived from a subject scheduled for administration of theinduced CTLs, or they may be derived from a different subject. When APCsor exosomes and CD8-positive T cells derived from a subject (donor)different from the subject scheduled for administration are used, thesubject of administration and the donor must have the identical HLAtype. For example, when a peptide having the amino acid sequenceselected from among SEQ ID NOs: 5, 28, 30 and 32 or a modified peptidethereof is used as peptides of the present invention, the HLA type inboth the subject of administration and the donor is preferably HLA-A11(more preferably HLA-A*1101). Alternatively, APCs or exosomes used inthe above-described methods are preferably APCs or exosomes that presenton their surface a complex of HLA-A11 (more preferably HLA-A*1101) and apeptide of the present invention (a peptide having the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30 and 32 or a modifiedpeptide thereof). In this case, the induced CTLs show a specificcytotoxic activity against cells that present a complex of HLA-A11 and apeptide of the present invention (for example, KOC1-expressingHLA-A11-positive cells). Alternatively, for example, when a peptidehaving the amino acid sequence selected from among SEQ ID NOs: 61, 62,63, 64, 67, 74, 77, 52, 79, 80 and 85 or a modified peptide thereof isused as peptides of the present invention, the HLA in the subject ofadministration and the donor is preferably both HLA-A33 (more preferablyHLA-A*3303). Alternatively, APCs or exosomes used in the above-describedmethods are preferably APCs or exosomes that present on their surface acomplex of HLA-A33 (more preferably HLA-A*3303) and a peptide of thepresent invention (a peptide having the amino acid sequence selectedfrom among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85 ora modified peptide thereof). In this case, the induced CTLs show aspecific cytotoxic activity against cells that present a complex ofHLA-A33 and a peptide of the present invention (for example,KOC1-expressing HLA-A33-positive cells). Alternatively, for example,when a peptide having the amino acid sequence selected from among SEQ IDNOs: 27, 30 and 52 or a modified peptide thereof is used as peptides ofthe present invention, the HLA type in both the subject ofadministration and the donor is preferably HLA-A03 (more preferablyHLA-A*0301). Alternatively, APCs or exosomes used in the above-describedmethods are preferably APCs or exosomes that present on their surface acomplex of HLA-A03 (more preferably HLA-A*0301) and a peptide of thepresent invention (a peptide having the amino acid sequence selectedfrom among SEQ ID NOs: 27, 30 and 52 or a modified peptide thereof). Inthis case, the induced CTLs show a specific cytotoxic activity againstcells that present a complex of HLA-A03 and a peptide of the presentinvention (for example, KOC1-expressing HLA-A03-positive cells).Alternatively, for example, when a peptide having the amino acidsequence selected from among SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41or a modified peptide thereof is used as peptides of the presentinvention, the HLA type in both the subject of administration and thedonor is preferably HLA-A01 (more preferably HLA-A*0101). Alternatively,APCs or exosomes used in the above-described methods are preferably APCsor exosomes that present on their surface a complex of HLA-A01 (morepreferably HLA-A*0101) and a peptide of the present invention (a peptidehaving the amino acid sequence selected from among SEQ ID NOs: 86, 87,90, 92, 46, 95 and 41 or a modified peptide thereof). In this case, theinduced CTLs show a specific cytotoxic activity against cells thatpresent a complex of HLA-A01 and a peptide of the present invention (forexample, KOC1-expressing HLA-A01-positive cells).

In another embodiment, the present invention also provides compositionsor pharmaceutical compositions for inducing CTLs, comprising at leastone active ingredient selected from below:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention; and

(d) an exosome that presents on its surface a peptide of the presentinvention.

In another embodiment, the present invention also provides use of anactive ingredient selected from below in the manufacture of compositionsor pharmaceutical compositions for inducing CTLs:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention; and

(d) an exosome that presents on its surface a peptide of the presentinvention.

Alternatively, the present invention further provides an activeingredient selected from below for use in inducing CTLs:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention; and

(d) an exosome that presents on its surface a peptide of the presentinvention.

Alternatively, the present invention further provides a method orprocess for manufacturing a composition or pharmaceutical compositionfor inducing CTLs, which is a method or process that comprises a step offormulating an active ingredient selected from below with apharmaceutically or physiologically acceptable carrier:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention; and

(d) an exosome that presents on its surface a peptide of the presentinvention.

In another embodiment, the present invention further provides a methodor process for manufacturing a composition or pharmaceutical compositionfor inducing CTLs, which is a method or process that comprises a step ofmixing an active ingredient selected from below with a pharmaceuticallyor physiologically acceptable carrier:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention; and

(d) an exosome that presents on its surface a peptide of the presentinvention.

XI. Methods of Inducing an Immune Response

The present invention further provides methods of inducing an immuneresponse against KOC1-expressing cancers. Applicable cancers includebladder cancer, cervical cancer, cholangiocellular cancer, chronicmyeloid leukemia (CML), colon cancer, rectum cancer, esophagus cancer,diffuse gastric cancer, non-small-cell lung cancer, small-cell lungcancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head andneck cancer, soft tissue tumor, testis cancer and such, but are notlimited thereto. It is preferable that the cancer expresses at least oneHLA selected from among HLA-A11, HLA-A33, HLA-A03 and HLA-A01.

The present invention further provides methods of inducing an immuneresponse against KOC1-expressing cancer cells. KOC1 is recognized to beoverexpressed in various types of cancers described above. Thus, when animmune response against KOC1-expressing cancer cells is induced,proliferation of the cancer cells is inhibited as a result. Accordingly,the present invention further provides methods of inhibitingproliferation of KOC1-expressing cancer cells. The methods of thepresent invention are suitable, in particular, for inhibitingproliferation of cancer cells expressing KOC1 and at least one HLAselected from among HLA-A11, HLA-A33, HLA-A03 and HLA-A01.

The methods of the present invention may comprise a step ofadministering a composition comprising any of the peptides of thepresent invention or a polynucleotide(s) encoding the peptide(s). Themethods of the present invention also contemplate administration of APCsor exosomes presenting any of the peptides of the present invention. Thedetails can be referred to the “IX. Pharmaceutical compositions”section, particularly portions describing regarding use of thepharmaceutical compositions of the present invention as vaccines. Inaddition, exosomes and APCs that can be used in the methods of thepresent invention for inducing an immune response are described indetail in “V. Exosomes”, “VI. Antigen-presenting cells (APCs)” and inItems (1) and (2) of “X. Methods of using peptides, exosomes, APCs andCTLs” described above.

In another embodiment, the present invention provides pharmaceuticalcompositions or vaccines for inducing an immune response againstKOC1-expressing cancers, wherein the pharmaceutical composition orvaccine comprises an active ingredient selected from below:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

Alternatively, the present invention also provides pharmaceuticalcompositions or vaccines for inducing an immune response againstKOC1-expressing cancer cells, wherein the pharmaceutical composition orvaccine comprises an active ingredient selected from below:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides pharmaceuticalcompositions or vaccines for inhibiting proliferation of KOC1-expressingcancer cells, wherein the pharmaceutical composition or vaccinecomprises an active ingredient selected from below:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

In another embodiment, the present invention provides use of an activeingredient selected from below in the manufacture of pharmaceuticalcompositions or vaccines for inducing an immune response againstKOC1-expressing cancers:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

Alternatively, the present invention also provides use of an activeingredient selected from below in the manufacture of pharmaceuticalcompositions or vaccines for inducing an immune response againstKOC1-expressing cancer cells:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

Alternatively, the present invention further provides use of an activeingredient selected from below in the manufacture of pharmaceuticalcompositions or vaccines for inhibiting proliferation of KOC1-expressingcancer cells:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents on its surface a peptide of the presentinvention;

(d) an exosome that presents on its surface a peptide of the presentinvention; and

(e) a CTL of the present invention.

The present invention further provides methods or processes formanufacturing pharmaceutical compositions that induce an immune responseagainst KOC1-expressing cancers, which is a method that may comprise astep of mixing or formulating a peptide or polynucleotide of the presentinvention with a pharmaceutically acceptable carrier.

Alternatively, the present invention provides methods for inhibitingproliferation of KOC1-expressing cancer cells or methods of inducing animmune response against KOC1-expressing cancers, which comprises a stepof administering to a subject vaccines or pharmaceutical compositionscomprising an active ingredient selected from below:

(a) a peptide of the present invention;

(b) a polynucleotide encoding a peptide of the present invention in anexpressible form;

(c) an APC that presents a peptide of the present invention on itssurface;

(d) an exosome that presents a peptide of the present invention on itssurface; and

(e) a CTL of the present invention.

In the context of the present invention, KOC1-expressing cancers can betreated by administering a peptide, a polynucleotide, an APC, an exosomeand/or a CTL of the present invention. Alternatively, an immune responseagainst KOC1-expressing cancers can be induced by administering apeptide, a polynucleotide, an APC, an exosome and/or a CTL of thepresent invention. Examples of such cancers include bladder cancer,cervical cancer, cholangiocellular cancer, chronic myeloid leukemia(CML), colon cancer, rectum cancer, esophagus cancer, diffuse gastriccancer, non-small-cell lung cancer, small-cell lung cancer, lymphoma,osteosarcoma, ovarian cancer, kidney cancer, head and neck cancer, softtissue tumor, testis cancer and such, but are not limited thereto.Further, an immune response against KOC1-expressing cancer cells can beinduced by administering a peptide, a polynucleotide, an APC, an exosomeand/or a CTL of the present invention. Therefore, before administering avaccine or pharmaceutical composition comprising an active ingredientdescribed above, it is preferable to confirm whether the level of KOC1expression at a diseased site in the subject to be treated is augmentedor not.

Thus, in one embodiment, the present invention provides a method oftreating a KOC1-expressing cancer in a patient in need of the cancertreatment, wherein the method comprises the steps below:

-   (i) measuring the level of KOC1 expression in a biological sample    collected from the diseased site of a subject with cancer;-   (ii) identifying a subject with KOC1-expressing cancer based on the    KOC1 expression level measured in (i); and-   (iii) administering to the subject with KOC1-expressing cancer at    least one ingredient selected from the group consisting of (a)    to (e) above.

Alternatively, the present invention further provides vaccines andpharmaceutical compositions comprising at least one active ingredientselected from the group consisting of (a) to (e) above foradministration to a subject with KOC1-expressing cancer. The presentinvention further provides a method of identifying or selecting asubject to be treated with at least one active ingredient selected fromthe group consisting of (a) to (e) above, wherein the method comprisesthe steps below:

-   (i) measuring the level of KOC1 expression in a biological sample    collected from the diseased site of a subject with cancer;-   (ii) identifying a subject with KOC1-expressing cancer based on the    KOC1 expression level measured in (i); and-   (iii) identifying or selecting the subject identified in (ii) as a    subject who may be treated with at least one active ingredient    selected from the group consisting of (a) to (e) above.

Biological samples collected from a subject for measuring the KOC1expression level in the above-described methods are not particularlylimited, and for example, tissue samples containing cancer cellscollected by biopsy or such can be preferably used. The KOC1 expressionlevel in a biological sample can be measured by known methods, and forexample, methods that detect transcription products of the KOC1 gene byprobes or PCR methods (for example, cDNA microarray method, Northernblot method, RT-PCR method or such), methods that detect translationproducts of the KOC1 gene by antibodies or such (for example, Westernblot method, immunostaining method or such), and such can be used.Further, biological samples may be blood samples, and in this case, theblood level of an antibody against KOC1 is measured, and the KOC1expression level at a diseased site may be assessed based on the bloodlevel. The blood level of an antibody against KOC1 or a fragment thereofcan be measured using known methods, and for example, enzyme immunoassay(EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA)and such using the KOC1 protein or a peptide of the present invention asan antigen can be used.

Normally, in tissues and cells that do not express KOC1, there is almostno detection of KOC1 transcription products and translation products.Thus, when a transcription product or a translation product of KOC1 isdetected in cancer cells or a tissue sample containing cancer cellscollected from a subject, one can determine that the subject's cancerexpresses KOC1. In blood samples of a subject that does not haveKOC1-expressing cancer, there is almost no detection of antibodiesagainst KOC1 or fragments thereof. Thus, when antibodies against KOC1 orfragments thereof are detected in a blood sample collected from asubject, one can determine that the subject's cancer expresses KOC1.Whether a subject's cancer expresses KOC1 or not may also be determinedby comparison with the measurement results of the same type ofbiological material collected from a non-cancerous site of the subjector the same type of biological material collected from a subject whodoes not have cancer (normal control sample). That is, in comparisonwith the level of the target of measurement in a normal control sample(normal control level), when the level in the biological sample of thetest subject is elevated, the subject's cancer is assessed to beexpressing KOC1. For example, when the amount of the target ofmeasurement detected is increased by at least 10% or higher incomparison with the normal control level, the subject's cancer may beassessed to be expressing KOC1. It is desirable that the amount of thetarget of measurement detected is increased by preferably 25% or higher,and more preferably 50% or higher than the normal control level.Further, the amount of a transcription product or a translation productof KOC1 detected may be evaluated by normalizing against the detectedamount of a known housekeeping gene such as beta-Actin,glyceraldehyde-3-phosphate dehydrogenase, or ribosomal protein P1.

In a preferred embodiment, it is preferable to confirm the HLA type ofthe subject before administering at least one active ingredient selectedfrom the group consisting of (a) to (e) above. For example, for thesubjects to be administered with an active ingredient in associationwith a peptide having the amino acid sequence selected from among SEQ IDNOs: 5, 28, 30 and 32, it is preferable to select HLA-A11-positivesubjects. For the subjects to be administered with an active ingredientin association with a peptide having the amino acid sequence selectedfrom among SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85, itis preferable to select HLA-A33-positive subjects. For the subjects tobe administered with an active ingredient in association with a peptidehaving the amino acid sequence selected from among SEQ ID NOs: 27, 30and 52, it is preferable to select HLA-A03-positive subjects. For thesubjects to be administered with an active ingredient in associationwith a peptide having the amino acid sequence selected from among SEQ IDNOs: 86, 87, 90, 92, 46, 95 and 41, it is preferable to selectHLA-A01-positive subjects.

The present invention further provides complexes of a peptide of thepresent invention and HLA. The complexes of the present inventiondescribed above may be monomers or multimers. When a complex of thepresent invention is a multimer, the number of polymerization is notparticularly limited, and it can be a multimer of any number ofpolymerization. Examples include a tetramer, pentamer, hexamer and such,but are not limited thereto. The multimers of the present invention alsoencompass dextramers (WO2002/072631) and streptamers (Knabel Metal., NatMed. 2002 June; 8(6): 631-7.). Complexes of a peptide of the presentinvention and HLA can be prepared according to known methods (forexample, Altman J D et al., Science. 1996, 274(5284): 94-6;WO2002/072631; WO2009/003492; Knabel M et al., Nat Med. 2002 June; 8(6):631-7, and such). The complexes of the present invention, for example,can be used in the quantification of CTLs specific to a peptide of thepresent invention. For example, a blood sample is collected from asubject administered with a pharmaceutical composition of the presentinvention, and CD4-negative cells are prepared after separation of PBMCsand contacted with a fluorescent dye-conjugated complex of the presentinvention. Then, the percentage of CTLs specific to a peptide of thepresent invention can be measured by flow cytometry analysis. Forexample, immune response-inducing effects by a pharmaceuticalcomposition of the present invention can be monitored by measuring thespecific CTLs against a peptide of the present invention before, duringand/or after administration of the pharmaceutical composition of thepresent invention.

XII. Antibodies

The present invention further provides antibodies that bind to thepeptide of the present invention. Preferable antibodies bindspecifically to a peptide of the present invention, but do not bind (orweakly bind) to one that is not the peptide of the present invention. Inanother embodiment, such an antibody may include an antibody thatrecognizes a peptide in the context of HLA molecules, i.e., an antibodythat binds to a peptide-MHC complex. The binding specificity of anantibody can be confirmed by inhibition assay. That is, if the bindingbetween an antibody to be analyzed and a full-length KOC1 polypeptide isinhibited in the presence of a peptide of the present invention, thisantibody is shown to specifically bind to the peptide of the presentinvention. Antibodies against peptides of the present invention can beused in assays of disease diagnosis and prognosis, as well as subjectselection for administration of the pharmaceutical compositions of thepresent invention and monitoring of the pharmaceutical compositions ofthe present invention.

The present invention also provides various immunological assays fordetecting and/or quantifying peptides of the present invention orfragments thereof. Such immunological assays include radioimmunoassay,immunochromatography, enzyme-linked immunosorbent assay (ELISA),enzyme-linked immunofluorescence assay (ELIFA) and such, without beinglimited thereto, and are performed within the scope of the variousimmunological assay formats well known in the art.

The antibodies of the present invention can be used in immunologicalimaging methods that can detect KOC1-expressing cancers, and examplesthereof include radioactive scintigraphic imaging using a labelledantibody of the present invention, without being limited thereto. Suchassay methods are used clinically in the detection, monitoring, andprognosis of KOC1-expressing cancers; and examples of such cancerinclude bladder cancer, cervical cancer, cholangiocellular cancer,chronic myeloid leukemia (CML), colon cancer, rectum cancer, esophaguscancer, diffuse gastric cancer, non-small-cell lung cancer, small-celllung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, headand neck cancer, soft tissue tumor, testis cancer and such, withoutbeing limited thereto.

The antibodies of the present invention can be used in any arbitraryform such as monoclonal antibodies or polyclonal antibodies, and mayfurther include anti-sera obtained by immunizing an animal such as arabbit with a peptide of the present invention, all classes ofpolyclonal antibodies and monoclonal antibodies, human antibodies, aswell as chimeric antibodies and humanized antibodies generated throughgene recombination.

The peptide of the present invention or a fragment thereof used as anantigen for obtaining antibodies can be obtained by chemical synthesisor genetic engineering techniques based on the amino acid sequencesdisclosed herein.

The peptide used as an immunizing antigen may be a peptide of thepresent invention or a fragment of a peptide of the present invention.Further, the peptide may be bound to or conjugated with a carrier forincreasing immunogenicity. Keyhole limpet hemocyanin (KLH) is well-knownas a carrier. Methods for binding KLH to a peptide are also well knownin the art.

Any mammal can be immunized with an antigen described above, and it ispreferable to consider the compatibility with the parent cell used incell fusion when generating a monoclonal antibody. Generally, animals ofthe order Rodentia, Lagomorpha or Primate can be used. Animals of theorder Rodentia include, for example, mice, rats and hamsters. Animals ofthe order Lagomorpha include, for example, rabbits. Animals of the orderPrimate include, for example, Catarrhini monkeys (old world monkeys)such as cynomolgus monkey (Macaca fascicularis), rhesus monkeys,hamadryas, and chimpanzee.

Methods of immunizing animals with an antigen are known in the art.Intraperitoneal injection and subcutaneous injection of an antigen arestandard methods for immunizing mammals. More specifically, an antigenis diluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS), physiological saline, or such. As needed, an antigensuspension solution can be administered to mammals after being mixedwith an appropriate amount of a standard adjuvant such as Freund'scomplete adjuvant and emulsified. Then, it is preferable to administerthe antigen mixed with an appropriate amount of a Freund's incompleteadjuvant several times every 4 to 21 days. A suitable carrier may beused for immunization. After the above immunization, the serum can beexamined by standard method with respect to increase in the quantity ofthe desired antibody.

Polyclonal antibodies against a peptide of the present invention can beprepared by collecting blood from mammals that have been confirmed withan increase in the serum level of the desired antibody afterimmunization, and separating the serum from blood by any conventionalmethod. A polyclonal antibody may be a polyclonal antibody-containingserum, or a polyclonal antibody-containing fraction may be isolated fromthe serum. Immunoglobulin G or M can be prepared from fractions thatrecognize only a peptide of the present invention by, for example, usingan affinity column conjugated with the peptide of the present invention,and then further purifying the fractions using a protein A or protein Gcolumn.

In order to prepare monoclonal antibodies, upon confirming an increasein the serum level of the desired antibody after immunization, immunecells are collected from the mammals and subjected to cell fusion.Immune cells used for cell fusion may be preferably obtained from thespleen. For the other parent cells fused with the above immune cells,for example, a mammalian myeloma cell, preferably a myeloma cell thathas acquired a property for drug selection of fusion cells can be used.

The above immune cells can be fused with myeloma cells by followingknown methods, for example, the method of Milstein et at (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Hybridomas obtained by cell fusion can be selected by culturing them ina standard selection medium such as the HAT medium (a medium containinghypoxanthine, aminopterin and thymidine). Cell culturing is continued inthe HAT medium for a sufficient period of time (for example, severaldays to several weeks) to allow death of all other cells (non-fusedcells) besides the desired hybridomas. Then, hybridoma cells producingthe desired antibody can be screened and cloned by performing a standardlimiting dilution.

In addition to the above methods of immunizing a non-human animal withan antigen for hybridoma preparation, human lymphocytes such as EBvirus-infected lymphocytes can be immunized in vitro with a peptide,cells expressing the peptide, or lysates thereof. Then, the immunizedlymphocytes can be fused with immortalized human-derived myeloma cellssuch as U266 to obtain hybridomas producing a desired human antibodycapable of binding to the peptide (JPS63-17688).

Next, the obtained hybridoma is transplanted into the abdominal cavityof a mouse, and the ascites is extracted. The obtained monoclonalantibody can be purified by, for example, ammonium sulfateprecipitation, protein A or protein G column, DEAE ion-exchangechromatography, or affinity column conjugated with the peptide of thepresent invention.

Alternatively, antibody-producing immune cells such as the immunizedlymphocytes can be immortalized by a cancer gene and used for thepreparation of monoclonal, antibodies.

The monoclonal antibodies obtained as such can also be prepared byrecombination using genetic engineering techniques (see, e.g.,Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies published inUnited Kingdom by MacMillan Publishers LTD (1990)). For example, anantibody-encoding DNA can be cloned from immune cells such asantibody-producing hybridoma or immunized lymphocytes and inserted intoa suitable vector, and then this is introduced into host cells toprepare a recombinant antibody. The present invention also providesrecombinant antibodies prepared as described above.

Further, the antibodies of the present invention may be antibodyfragments or modified antibodies, as long as they bind to the peptidesof the present invention. For example, it is desirable that the antibodyfragment contains an antigen-binding site(s) of the antibodies.Specifically, the antibody fragments may be Fab, F(ab′)₂, Fv, or asingle chain Fv(scFv) in which Fv fragments derived from an H chain andan L chain are linked with a suitable linker (Huston et al., Proc NatiAcad Sci USA 85: 5879-83 (1988)). More specifically, antibody fragmentsmay be generated by treating an antibody with an enzyme such as papainor pepsin. Alternatively, a gene encoding an antibody fragment may beconstructed, inserted into an expression vector, and expressed in anappropriate host cell (see, e.g., Co et al., J Immunol 152: 2968-76(1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989);Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi,Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

Antibodies may be modified by conjugation with various molecules such aspolyethyleneglycol (PEG). The present invention provides such modifiedantibodies. Modified antibodies can be obtained by chemically modifyingthe antibodies. These modification methods are conventional in the art.

Alternatively, the antibodies of the present invention can be obtainedas chimeric antibodies of a non-human antibody-derived variable regionand a human antibody-derived constant region, or as humanized antibodiescomprising a non-human antibody-derived complementarity determiningregion (CDR) and a human antibody-derived framework region (FR) andconstant region. Such antibodies can be prepared according to knowntechniques. Humanization can be carried out by substituting a humanantibody sequence(s) with a corresponding non-human antibody CDRsequence(s) (see, e.g., Verhoeyen et al., Science 239: 1534-6 (1988)).Thus, such humanized antibodies are chimeric antibodies in which thesubstantially less than an intact human variable domain has beensubstituted with a corresponding sequence from a non-human species.

Intact human antibodies comprising a human variable region in additionto the human framework and constant regions can also be used. Suchantibodies can be generated using various techniques known in the art.For example, in vitro methods include use of recombinant libraries ofhuman antibody fragments presented on bacteriophages (for example,Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, humanantibodies can also be generated by introducing human immunoglobulingene loci into transgenic animals, for example, mice, in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in, for example, U.S. Pat. Nos.6,150,584, 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and5,661,016.

Antibodies obtained as described above may be purified to homogeneity.For example, antibody separation and purification can be performedaccording to separation methods and purification methods used forgeneral proteins. For example, an antibody can be separated and isolatedby appropriately selecting and combining use of column chromatographiessuch as affinity chromatography, filter, ultrafiltration, salting-out,dialysis, SDS-polyacrylamide gel electrophoresis and isoelectricfocusing electrophoresis (Antibodies: A Laboratory Manual. Ed Harlow andDavid Lane, Cold Spring Harbor Laboratory (1988)), but are not limitedthereto. Protein A column and protein G column can be used as theaffinity column. Exemplary protein A columns to be used include, forexample, Hyper D, POROS and Sepharose F. F. (Pharmacia).

Besides affinity chromatography, exemplary chromatography includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reversed-phase chromatography, adsorption chromatography andsuch (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). The chromatography procedures can becarried out by liquid-phase chromatography such as HPLC and FPLC.

The antigen-binding activity of an antibody of the present invention canbe measured, for example, by using absorbance measurement, enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay(RIA), and/or immunofluorescence (IF). In the case of ELISA, an antibodyof the present invention is immobilized onto a plate, a peptide of thepresent invention is applied to the plate, and then a sample containingthe desired antibody, such as culture supernatant of antibody-producingcells or purified antibodies, is applied. Next, a secondary antibodythat recognizes the primary antibody and is labelled with an enzyme suchas alkaline phosphatase is applied and the plate is incubated. Then,after washing, an enzyme substrate such as p-nitrophenyl phosphate isapplied to the plate, and the antigen-binding activity of the sample isevaluated by measuring absorbance. To assess the binding activity of anantibody, peptide fragments such as C-terminal or N-terminal fragmentsmay be used as an antigen. BIAcore (Pharmacia) may be used to evaluatethe activity of an antibody of the present invention.

It is possible to detect or measure a peptide of the present inventionusing the above methods, by exposing an antibody of the presentinvention to a sample assumed to contain the peptide of the presentinvention, and detecting or measuring an immune complex formed betweenthe antibody and the peptide.

For example, an antibody of the present invention can be used to detecta peptide of the present invention present in the blood sample (forexample, serum sample) of a subject. Alternatively, an antibody of thepresent invention present in the blood sample (for example, serumsample) of a subject can also be detected using a peptide of the presentinvention. The result of measuring a peptide of the present invention oran antibody of the present invention in the blood sample of a subjectcan be utilized to the subject selection for administration of thepharmaceutical compositions of the present invention or monitoring ofthe efficacy of the pharmaceutical compositions of the presentinvention. In addition, it has been reported that patients having anantibody against a peptide administered as vaccine may have highresponsiveness to the vaccine. Therefore, the peptide of the presentinvention can be utilized as an immunoassay antigen for selecting apatient with high responsiveness when the peptide is administered as avaccine using an antibody of the patient as an index.

XIII. Vectors and Host Cells

The present invention provides vectors comprising a polynucleotideencoding a peptide of the present invention and host cells introducedwith the vectors. A vector of the present invention may be used to keepa polynucleotide of the present invention in a host cell, to express apeptide of the present invention in a host cell, or to administer apolynucleotide of the present invention for gene therapy.

When E. coli is a host cell and a vector is amplified and produced in alarge amount in E. coli (for example, JM109, DH5-alpha, HB101 orXL1-Blue), the vector needs to have a “replication origin” foramplification in E. coli and a marker gene for selection of transformedE. coli (for example, a drug resistance gene selected by a drug such asampicillin, tetracycline, kanamycin, chloramphenicol). For example, theM13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Scriptand such can be used. In addition, pGEM-T, pDIRECT and pT7 can be usedfor cloning as well as the above vectors. When a vector is used in theproduction of a peptide of the present invention, an expression vectorcan be used. For example, an expression vector for expression in E. colineeds to have the above features for amplification in E. coli. When E.coli such as JM109, DH5-alpha, HB101 or XL1-Blue are used as a hostcell, the vector needs to have a promoter, for example, lacZ promoter(Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1989)), araBpromoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or thelike, that can efficiently express the desired gene in E. coli. In thatrespect, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP andpET (in this case, the host is preferably BL21 which expresses T7 RNApolymerase), for example, can be used instead of the above vectors.Additionally, the vector may contain a signal sequence for peptidesecretion. An exemplary signal sequence that directs the peptide to besecreted to the periplasm of the E. coli is the pelB signal sequence(Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing thevectors into the target host cells include, for example, the calciumchloride method and the electroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic AcidsRes 18(17): 5322 (1990)), pEF, pCDM8), expression vectors derived frominsect cells (for example, “Bac-to-BAC baculovirus expression system”(GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g.,pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV,pMV, pAdexLcw), expression vectors derived from retroviruses (e.g.,pZIpneo), expression vectors derived from yeast (e.g., “PichiaExpression Kit” (Invitrogen), pNV11, SP-Q01) and expression vectorsderived from Bacillus subtilis (e.g., pPL608, pKTH50) can be used forproducing the polypeptide of the present invention.

In order to express the vector in animal cells such as CHO, COS orNIH3T3 cells, the vector needs to carry a promoter necessary forexpression in such cells, for example, the SV40 promoter (Mulligan etal., Nature 277: 108 (1979)), the MMLV-LTR promoter, the EFI-alphapromoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMVpromoter and the like, and preferably a marker gene for selectingtransformants (for example, a drug resistance gene selected by a drug(e.g., neomycin, G418)). Examples of known vectors with thesecharacteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV,pOPRSV and pOP13.

The embodiments of the present invention are exemplified below based onthe above explanation; however, the present invention is not limited tothese embodiments.

[1] A peptide of less than 15 amino acids having cytotoxic T cell(CTL)-inducing ability, which comprises the amino acid sequence selectedfrom the group of:

-   -   (a) the amino acid sequence selected from the group consisting        of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52,        79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41; and    -   (b) the amino acid sequence in which one, two or several amino        acids are substituted, deleted, inserted and/or added to the        amino acid sequence selected from the group consisting of SEQ ID        NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85,        27, 86, 87, 90, 92, 46, 95 and 41.        [2] The peptide of [1], which is selected from the group        consisting of (i) to (iv) below:    -   (i) a peptide comprising the amino acid sequence in which one or        more substitution(s) selected from the group consisting of (a)        to (d) below is introduced into the amino acid sequence selected        from the group consisting of SEQ ID NOs: 5, 28, 30 and 32:        -   (a) the second amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            threonine, valine, isoleucine, leucine, phenylalanine and            tyrosine;        -   (b) the third amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            leucine, phenylalanine, tyrosine, isoleucine and alanine;        -   (c) the seventh amino acid from the N terminus is            substituted with an amino acid selected from the group            consisting of leucine, isoleucine, tyrosine, valine and            phenylalanine; and        -   (d) the C-terminal amino acid is substituted with an amino            acid selected from the group consisting of lysine and            arginine;    -   (ii) a peptide comprising the amino acid sequence in which one        or more substitution(s) selected from the group consisting        of (a) to (c) below is introduced into the amino acid sequence        selected from the group consisting of SEQ ID NOs: 61, 62, 63,        64, 67, 74, 77, 52, 79, 80 and 85:        -   (a) the first amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            aspartic acid and glutamic acid;        -   (b) the second amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            phenylalanine, tyrosine, alanine, isoleucine, leucine and            valine; and        -   (c) the C-terminal amino acid is substituted with an amino            acid selected from the group consisting of arginine and            lysine;    -   (iii) a peptide comprising the amino acid sequence in which one        or more substitution(s) selected from the group consisting        of (a) to (b) below is introduced into the amino acid sequence        selected from the group consisting of SEQ ID NOs: 27, 30 and 52:        -   (a) the second amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            leucine, methionine, valine, alanine, isoleucine, serine and            threonine; and        -   (b) the C-terminal amino acid is substituted with an amino            acid selected from the group consisting of arginine, lysine,            tyrosine and phenylalanine; and    -   (iv) a peptide comprising the amino acid sequence in which one        or more substitution(s) selected from the group consisting        of (a) to (c) below is introduced into the amino acid sequence        selected from the group consisting of SEQ ID NOs: 86, 87, 90,        92, 46, 95 and 41:        -   (a) the second amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            threonine and serine;        -   (b) the third amino acid from the N terminus is substituted            with an amino acid selected from the group consisting of            aspartic acid and glutamic acid; and        -   (c) the C-terminal amino acid is substituted with tyrosine.            [3] The peptide of [1], which consists of the amino acid            sequence selected from the group consisting of SEQ ID NOs:            5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85,            27, 86, 87, 90, 92, 46, 95 and 41.            [4] A polynucleotide, which encodes the peptide of any one            of [1] to [3].            [5] A composition comprising a pharmaceutically acceptable            carrier and at least one ingredient selected from the group            consisting of (a) to (e) below:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [6] The composition of [5], which is a composition for inducing        a CTL(s), wherein the ingredient is at least one ingredient        selected from the group consisting of (a) to (d) below:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen; and    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen.        [7] The composition of [5], which is a pharmaceutical        composition.        [8] The composition of [7], which is for one or more uses        selected from the group consisting of (i) cancer treatment, (ii)        cancer prevention (prophylaxis) and (iii) prevention        (prophylaxis) of postoperative cancer recurrence.        [9] The composition of [7], which is for inducing an immune        response against cancer.        [10] The composition of [8] or [9], wherein the cancer is        selected from the group consisting of bladder cancer, cervical        cancer, cholangiocellular cancer, chronic myeloid leukemia        (CML), colon cancer, rectum cancer, esophagus cancer, diffuse        gastric cancer, non-small-cell lung cancer, small-cell lung        cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer,        head and neck cancer, soft tissue tumor and testis cancer.        [11] The composition of any one of [5] to [10], which is        formulated for administration to a subject positive for at least        one HLA selected from the group consisting of HLA-A11, HLA-A33,        HLA-A03 and HLA-A01.        [12] A method of inducing an APC(s) having CTL-inducing ability,        which comprises a step selected from the group consisting of (a)        and (b) below:    -   (a) contacting an APC(s) with the peptide of any one of [1] to        [3] in vitro, ex vivo or in vivo; and    -   (b) introducing a polynucleotide encoding the peptide of any one        of [1] to [3] into an APC(s).        [13] A method of inducing a CTL(s), which comprises a step        selected from the group consisting of (a) to (c) below:    -   (a) co-culturing a CD8-positive T cell(s) with an APC(s) that        presents on its surface a complex of an HLA antigen and the        peptide of any one of [1] to [3];    -   (b) co-culturing a CD8-positive T cell(s) with an exosome(s)        that presents on its surface a complex of an HLA antigen and the        peptide of any one of [1] to [3]; and    -   (c) introducing into a CD8-positive T cell(s) a polynucleotide        encoding each subunit of a T cell receptor (TCR) capable of        binding to the peptide of any one of [1] to [3] presented by an        HLA antigen on a cell surface.        [14] An APC that presents on its surface a complex of an HLA        antigen and the peptide of any one of [1] to [3].        [15] The APC of [14], which is induced by the method of [12].        [16] A CTL that targets the peptide of any one of [1] to [3].        [17] The CTL of [16], which is induced by the method of [13].        [18] A method of inducing an immune response against cancer,        which comprises administering to a subject at least one        ingredient selected from the group consisting of (a) to (e)        below:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [19] A method of treating and/or preventing cancer, and/or        preventing postoperative recurrence thereof, which comprises        administering to a subject at least one ingredient selected from        the group consisting of (a) to (e) below:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [ 1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [20] An antibody that binds to the peptide of any one of [1] to        [3].        [21] A method of screening for a peptide having CTL-inducing        ability, which comprises the steps of:    -   (a) generating candidate sequences consisting of an amino acid        sequence in which one, two or several amino acid residues are        substituted, deleted, inserted and/or added to an original amino        acid sequence consisting of the amino acid sequence selected        from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62,        63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95        and 41;    -   (b) selecting from among the candidate sequences generated in        (a), a candidate sequence that does not have significant        homology (sequence identity) with any known human gene product        other than KOC1;    -   (c) contacting an APC(s) with a peptide consisting of the        candidate sequence selected in (b);    -   (d) contacting the APC(s) of (c) with a CD8-positive T cell(s);        and    -   (e) selecting a peptide having an equal to or higher        CTL-inducing ability than that of a peptide consisting of the        original amino acid sequence.        [22] Use of at least one ingredient selected from the group        consisting of (a) to (e) below in the manufacture of a        composition for inducing an immune response against cancer:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [23] Use of at least one ingredient selected from the group        consisting of (a) to (e) below in the manufacture of a        pharmaceutical composition for treating and/or preventing        cancer, and/or preventing postoperative recurrence thereof:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [24] Use of at least one ingredient selected from the group        consisting of (a) to (e) below for inducing an immune response        against cancer:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [25] Use of at least one ingredient selected from the group        consisting of (a) to (e) below for treating and/or preventing        cancer and/or preventing postoperative recurrence thereof:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [26] A method of inducing cytotoxic activity against a        KOC1-expressing cell(s), which comprises a step of administering        to a subject at least one ingredient selected from the group        consisting of (a) to (e) below:    -   (a) one or more types of peptides of any one of [1] to [3];    -   (b) one or more types of polynucleotides encoding the peptide(s)        of any one of [1] to [3] in an expressible form;    -   (c) an antigen-presenting cell (APC) that presents on its cell        surface a complex of the peptide of any one of [1] to [3] and an        HLA antigen;    -   (d) an exosome that presents on its cell surface a complex of        the peptide of any one of [1] to [3] and an HLA antigen; and    -   (e) a CTL that targets the peptide of any one of [1] to [3].        [27] A freeze-dried formulation comprising one or more types of        peptides of any one of [1] to [3].        [28] A pharmaceutical composition, which is prepared by a method        that comprises dissolving one or more types of peptides of any        one of [1] to [3] in a water-soluble carrier, and performing        filtration sterilization.        [29] A filtration-sterilized aqueous solution, which is an        aqueous solution that comprises one or more types of peptides of        any one of [1] to [3] and a water-soluble carrier.        [30] An emulsion comprising one or more types of peptides of any        one of [1] to [3], a water-soluble carrier and an oil adjuvant.        [31] A kit comprising a container that houses the pharmaceutical        composition of any one of [7] to [11] and a container that        houses an adjuvant.        [32] A kit comprising a container that stores a freeze-dried        formulation comprising the peptide of any one of [1] to [3], a        container that stores an adjuvant, and a container that stores a        re-dissolving solution the freeze-dried formulation.

The present invention is explained herein in detail with reference toits specific embodiments. However, it should be understood that theabove explanation is in fact an illustrative and explanatoryexplanation, and is intended to explain the present invention andpreferred embodiments thereof. Through routine experimentation, oneskilled in the art will readily recognize that various changes andmodifications can be made therein without departing from the spirit andscope of the present invention. Thus, the present invention is notconfined to the above explanation, but is intended to be defined by theappended claims and equivalents thereto.

Hereinbelow, the present invention is described in more detail withreference to the Examples. Nevertheless, while the following materials,method and Examples may serve to assist one of ordinary skill in makingand using certain embodiments of the present invention, there are onlyintended to illustrate aspects of the present invention and thus in noway to limit the scope of the present invention. One of ordinary skillin the art can use methods and materials similar or equivalent to thosedescribed herein in the practice or testing of the present invention.

All prior art documents cited herein are incorporated by reference inthe present specification.

EXAMPLES Example 1

Materials and Methods

Cell Lines

C1R, an HLA-A- and HLA-B-negative human B lymphoblastoid cell line, andCOST, an African green monkey kidney cell line, were purchased fromATCC.

Generation of Stimulator Cells with Stable HLA-A*1101 Expression

C1R (C1R-A11) that stably expresses HLA-A*1101 was used as a stimulatorcell. A cDNA encoding the open-reading frame of HLA-A*1101 was amplifiedby PCR and cloned into an expression vector. C1R cells were transfectedwith the expression vector, and then selected using G418 (Invitrogen)for two weeks. The G418-selected cells were seeded into wells containingG418-added culture medium in a 96-well plate, and further cultured for30 days. The exogenous HLA-A*1101 expression in C1R cells was verifiedby flow cytometric analysis.

Selection of Candidate KOC1-Derived Peptides

KOC1-derived 9mer and 10 mer peptides that bind to the HLA-A*1101molecule were predicted using the binding prediction server “NetMHC 3.2”(www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003November, 62(5): 378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17; Bioinformatics. 2004 Jun. 12: 20(9): 1388-97).

Peptide Synthesis

The peptides were synthesized by Biosynthesis (Lewisville, Tex.)according to a standard solid-phase synthesis method, and purified byreversed phase high-performance liquid chromatography (HPLC). Thepeptides were analyzed for their purity (>90%) and identity byanalytical HPLC and mass spectrometry, respectively. The peptides weredissolved in dimethylsulfoxide at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as theantigen-presenting cell to induce a cytotoxic T lymphocyte (CTL)response against peptides presented on human leukocyte antigens (HLAs).As described in the other sections, DCs were generated in vitro(Nakahara S et al., Cancer Res 2003, 63(14): 4112-8). Specifically,peripheral-blood mononuclear cells isolated from healthy volunteers(HLA-A*1101-positive) with the Ficoll-Paque plus solution (Pharmacia)were separated by attaching to plastic tissue culture dishes (BectonDickinson) and concentrated as a monocyte fraction. Themonocyte-concentrated population was cultured in the presence of 1000IU/ml granulocyte macrophage colony-stimulating factor (R&D System) and1000 IU/ml interleukin(IL)-4 (R&D System) in an AIM-V medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). Afterseven days of culturing, the cytokine-induced DCs were pulsed with 20micro-g/ml each of the synthesized peptides in an AIM-V medium at 37degrees C. for three hours in the presence of 3 micro-g/ml beta2-microglobulin. The generated cells appeared to express on their cellsurface DC-associated molecules such as CD80, CD83, CD86 and HLA classII (data not shown). Next, these peptide-pulsed DCs were inactivated, byX-ray irradiation (20 Gy), and mixed in a 1:20 ratio with autologousCD8⁺ T cells obtained by positive selection using the CD8 PositiveIsolation Kit (Dynal). These culture products were seeded in a 48-wellplate (Corning). Each well was made to contain 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8⁺ T cells and 10 ng/ml IL-7 (R&D System) in 0.5 ml of theAIM-V/2% AS medium. Three days later, these culture products were addedwith a final concentration of 20 IU/ml IL-2 (CHIRON). On day 7 and day14, T cells were further stimulated with peptide-pulsed autologous DCs.The DCs were prepared every time by the same method as above. On day 21,after the third peptide stimulation, CTLs were examined against thepeptide-pulsed C1R-A11 by a human interferon (IFN)-gamma enzyme-linkedimmunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al.,Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006,97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

CTL Propagation Procedure

CTLs were propagated in culture using methods similar to those disclosedby Riddell et al. (Walter E A et al., N Engl J Med 1995, 333(16):1038-44; Riddell S R et al., Nat Med 1996, 2(2): 216-23). The CTLs wereco-cultured in a total of 25 ml AIM-V medium containing 5% AS (AIM-V/5%AS) and 40 ng/ml anti-CD3 antibody with two types of Mitomycin C-treatedhuman B lymphoblastoid cell lines at 5×10⁶ cells/flask. One day afterbeginning of the culturing, 120 IU/ml IL-2 was added to the culture. Ondays 5, 8 and 11, a fresh AIM-V/5% AS medium containing 30 IU/ml IL-2was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al.,Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006,97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

Establishment of CTL Clones

Dilution of CTLs was carried out to make 0.3, 1 and 3 cells/well in 96round-bottomed microtiter plates (Nalge Nunc International). The CTLswere co-cultured with two types of Mitomycin C-treated human Blymphoblastoid cell lines at 1×10⁴ cells/well in a total of 150micro-1/well AIM-V/5% AS medium with 30 ng/ml anti-CD3 antibody and 125IU/ml IL-2. Ten days later, IL-2 was added to the medium at 50micro-1/well to reach a final concentration of 125 IU/ml. On day 14, theCTL activity was tested, and the CTL clones were propagated using thesame method as described above (Uchida N et al., Clin Cancer Res 2004,10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; WatanabeT et al., Cancer Sci 2005, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, an IFN-gamma ELISPOT assay and anIFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed.Specifically, peptide-pulsed C1R-A11 (1×10⁴ cells/well) was prepared asthe stimulator cell. The induced CTLs, i.e., CTL lines and CTL clones,were used as the responder cell. The IFN-gamma ELISPOT assay andIFN-gamma ELISA were performed according to the manufacturer's manual.

Establishment of Target Cells Forcibly Expressing a Target Gene andHLA-A*1101

A cDNA encoding the open-reading frame of a target gene or HLA-A*1101was amplified by PCR. The PCR-amplified product was cloned into anexpression vector. Either or both of the target gene-expressing vectorand the HLA-A*1101-expressing vector were transfected into COST, whichis a cell line negative for the target gene and HLA, using Lipofectamine2000 (Invitrogen) following the manufacturer's recommended protocol. Twodays after transfection, the transfected cells were harvested usingversene (Invitrogen), and used as the target cell for CTL activity assay(5×10⁴ cells/well).

Results

Prediction of KOC1-Derived HLA-A*1101-Binding Peptides

Tables 1a and 1b show KOC1-derived 9mer peptides and 10 mer peptidesthat have been predicted to bind to HLA-A*1101 in the descending orderof binding affinity. A total of 60 peptides that potentially have anHLA-A*1101-binding ability was selected and investigated to determineepitope peptides.

TABLE 1a HLA-A11-binding 9mer peptides   derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 258 KSILEIMHK   16  1 121VVNVTYSSK   18  2 465 KAQGRIYGK   32  3 517 SSAEVVVPR   45  4 415LSVGAIIGK   76  5  28 PVSGPFLVK   85  6  52 KAIEALSGK   92  7 142FQLENFTLK  150  8 215 GATIRNITK  293  9 338 KAEEEIMKK  297 10 497FAAGRVIGK  405 11  15 PSDLESIFK  451 12 536 VVKITGHFY  757 13 479FVSPKEEVK  791 14 493 RVPSFAAGR 1100 15 272 KFTEEIPLK 1108 16 226QSKIDVHRK 1125 17 330 KGNVETCAK 1835 18 544 YACQVAQRK 1841 19 220NITKQTQSK 2617 20 301 KIEQDTDTK 2631 21 553 IQEILTQVK 3008 22 182QGSPGSVSK 3293 23 559 QVKQHQQQK 3313 24  58 SGKIELHGK 4377 25 205QFVGAIIGK 4472 26

TABLE 1b HLA-A11-binding 10mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 120 AVVNVTYSSK   13 27 414ALSVGAIIGK   33 28 456 ITGPPEAQFK   73 29 204 TQFVGAIIGK   86 30  73SVPKRQRIRK  103 31  14 APSDLESIFK  152 32 183 GSPGSVSKQK  165 33 496SFAAGRVIGK  168 34 516 LSSAEVVVPR  169 35 552 KIQEILTQVK  214 36 431LSRFAGASIK  342 37 181 RQGSPGSVSK  368 38 124 VTYSSKDQAR  437 39 225TQSKIDVHRK  478 40 535 VVVKITGHFY  678 41 224 QTQSKIDVHR  711 42  57LSGKIELHGK  904 43 141 GFQLENFTLK  911 44 568 ALQSGPPQSR 1065 45 312ISPLQELTLY 1262 46 441 IAPAEAPDAK 1570 47  27 IPVSGPFLVK 1622 48 570QSGPPQSRRK 1777 49  95 EVLDSLLVQY 2124 50 336 CAKAEEEIMK 2194 51 281ILAHNNFVGR 2264 52 466 AQGRIYGKIK 2595 53 160 AQQNPLQQPR 2689 54 420IIGKQGQHIK 2763 55 558 TQVKQHQQQK 2862 56 257 CKSILEIMHK 3467 57 291LIGKEGRNLK 3795 58 263 IMHKEAQDIK 4083 59 569 LQSGPPQSRR 4107 60 Startposition indicates the number of amino acid residue from the N-terminusof KOC1. The dissociation constant [Kd (nM)] is derived from“NetMHC3.2”.Induction of CTLs by the Predicted KOC1-Derived HLA-A*1101-RestrictedPeptides

CTLs against the KOC1-derived peptides were generated according to theprotocol described in “Materials and methods”. The peptide-specific CTLactivity was measured by an IFN-gamma ELISPOT assay (FIG. 1). Incomparison with the control, CTLs in Well #6 with KOC1-A11-9-415 (SEQ IDNO: 5) (a), Well #4 with KOC1-A11-10-414 (SEQ ID NO: 28) (b), Well #5with KOC1-A11-10-204 (SEQ ID NO: 30) (c), and Well #8 withKOC1-A11-10-14 (SEQ ID NO: 32) (d) showed potent IFN-gamma production.Meanwhile, despite that other peptides shown in Tables 1a and 1bpotentially have an HLA-A*1101-binding activity, specific CTL activitywas not detected as a result of stimulation by those peptides. Anexample of typical negative data is that specific IFN-gamma productionwas not observed from CTLs stimulated with KOC1-A11-9-258 (SEQ ID NO: 1)(e). As a result, four types of KOC1-derived peptides were selected aspeptides capable of inducing potent CTLs.

Establishment of CTL Lines and Clones Against the KOC1-DerivedHLA-A*1101-Restricted Peptides

CTL lines were established by propagating CTLs in Well #6 withKOC1-A11-9-415 (SEQ ID NO: 5) (a), Well #5 with KOC1-A11-10-204 (SEQ IDNO: 30) (b), and Well #8 with KOC1-A11-10-14 (SEQ ID NO: 32) (c), whichshowed peptide-specific CTL activity in the IFN-gamma ELISPOT assay. TheCTL activity of these CTL lines was measured by IFN-gamma ELISA (FIG.2). These CTL lines showed potent IFN-gamma production against targetcells pulsed with the respective peptides, in comparison with targetcells that have not been pulsed with the peptides. Further, CTL cloneswere established from the CTL lines by limiting dilution as described inthe “Materials and methods” section above, and IFN-gamma production fromthe CTL clones against peptide-pulsed C1R-A11 was measured by IFN-gammaELISA. Potent IFN-gamma production was observed in CTL clones stimulatedwith KOC1-A11-9-415 (SEQ ID NO: 5) (a) and KOC1-A11-10-14 (SEQ ID NO:32) (b) (FIG. 3).

Specific CTL Activity Against Target Cells Expressing KOC1 andHLA-A*1101

The CTL clone established against KOC1-A11-10-14 (SEQ ID NO: 32) wasinvestigated for its ability to recognize target cells expressing KOC1and the HLA-A*1101 molecule. COS7 cells transfected with bothfull-length KOC1 and the HLA-A*1101 gene (a specific model of targetcells expressing KOC1 and the HLA-A*1101 gene) were prepared as thetarget cell. COS7 cells transfected with either full-length KOC1 orHLA-A*1101 were prepared as the control. The KOC1-A11-10-14 (SEQ ID NO:32)-stimulated CTL clone demonstrated a potent CTL activity against COS7cells expressing both KOC1 and HLA-A*1101 (FIG. 4). On the other hand, asignificant specific CTL activity was not detected against the controlcells. These data clearly proved that KOC1-A11-10-14 (SEQ ID NO: 32) isa peptide generated from endogenous processing of KOC1, and is presentedon target cells with the HLA-A*1101 molecule and recognized by CTLs.These results demonstrated the possibility that KOC1-A11-10-14 (SEQ IDNO: 32) may be suitable as a cancer vaccine for patients having aKOC1-expressing cancer.

Homology Analysis of Antigen Peptides

CTLs stimulated with KOC1-A11-9-415 (SEQ ID NO: 5), KOC1-A11-10-414 (SEQID NO: 28), KOC1-A11-10-204 (SEQ ID NO: 30), or KOC1-A11-10-14 (SEQ IDNO: 32) demonstrated a significant specific CTL activity. These resultsmay be due to the fact that the KOC1-A11-9-415 (SEQ ID NO: 5),KOC1-A11-10-414 (SEQ ID NO: 28), KOC1-A11-10-204 (SEQ ID NO: 30), andKOC1-A11-10-14 (SEQ ID NO: 32) sequences are homologous to peptidesderived from other molecules known for sensitizing the human immunesystem. In order to exclude this possibility, homology analysis wasperformed by querying these peptide sequences using the BLAST algorithm(blast.ncbi.nlm.nih.gov/Blast.cgi). This result showed that there is nosequence having a significant homology with the KOC1-A11-9-415 (SEQ IDNO: 5), KOC1-A11-10-414 (SEQ ID NO: 28), KOC1-A11-10-204 (SEQ ID NO:30), and KOC1-A11-10-14 (SEQ ID NO: 32) sequences. On the other hand,KOC1-A11-10-204 (SEQ ID NO: 30) is identical to a peptide sequenceidentified in IMP-2 which is another IMP family. It has been previouslyreported that IMP-2 is not expressed in normal organs except testis andfetal liver (Hammer N A et al., Reproduction. 2005; 130(2): 203-12). Itis involved in the proliferation of glioblastoma cancer stem cells(Janiszewska M et al., Genes Dev. 2012; 26(17): 1926-44). IMP-2 ispromising as a target antigen for cancer immunotherapy. Therefore, tothe knowledge of the present inventors, there is almost no possibilitythat these peptides would elicit an unintended immune response againstother unrelated molecules. In conclusion, novel KOC1-derivedHLA-A11-restricted epitope peptides were identified. It was demonstratedthat the KOC1-derived epitope peptides are applicable for cancerimmunotherapy.

Example 2

Materials and Methods

Cell Lines

C1R, an HLA-A- and HLA-B-negative human B lymphoblastoid cell line, andCOST, an African green monkey kidney cell line, were purchased fromATCC.

Generation of Stimulator Cells with Stable HLA-A*3303 Expression

C1R (C1R-A33) that stably expresses HLA-A*3303 was used as a stimulatorcell. A cDNA encoding the open-reading frame of HLA-A*3303 was amplifiedby PCR and cloned into an expression vector. C1R cells were transfectedwith the expression vector, and then selected using G418 (Invitrogen)for two weeks. The G418-selected cells were seeded into wells containingG418-added culture medium in a 96-well plate, and further cultured for30 days. The exogenous HLA-A*3303 expression in C1R cells was verifiedby flow cytometric analysis.

Selection of Candidate KOC1-Derived Peptides

KOC1-derived 9mer and 10 mer peptides that bind to the HLA-A*3303molecule were predicted using the binding prediction server“NetMHCpan2.8” (www.cbs.dtu.dk/services/NetMHCpan/) (Nielsen et al.,PLoS One. 2007; 29; 2(8): e796; Hoof et al., Immunogenetics. 2009;61(1): 1-13).

Peptide Synthesis

These peptides were synthesized by Biosynthesis (Lewisville, Tex.)according to a standard solid-phase synthesis method, and purified byreversed phase high-performance liquid chromatography (HPLC). Thepeptides were analyzed for their purity (>90%) and identity byanalytical HPLC and mass spectrometry, respectively. The peptides weredissolved in dimethylsulfoxide at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as theantigen-presenting cell to induce a cytotoxic T lymphocyte (CTL)response against peptides presented on human leukocyte antigens (HLAs).As described in the other sections, DCs were generated in vitro(Nakahara S et al., Cancer Res 2003, 63(14): 4112-8). Specifically,peripheral-blood mononuclear cells isolated from healthy volunteers(HLA-A*3303-positive) with the Ficoll-Paque plus solution (Pharmacia)were separated by attaching to plastic tissue culture dishes (BectonDickinson) and concentrated as a monocyte fraction. Themonocyte-concentrated population was cultured in the presence of 1000IU/ml granulocyte macrophage colony-stimulating factor (R&D System) and1000 IU/ml interleukin(IL)-4 (R&D System) in an AIM-V medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). Afterseven days of culturing, the cytokine-induced DCs were pulsed with 20micro-g/ml each of the synthesized peptides in an AIM-V medium at 37degrees C. for three hours in the presence of 3 micro-g/ml beta2-microglobulin. The generated cells appeared to express on their cellsurface DC-associated molecules such as CD80, CD83, CD86 and HLA classII (data not shown). Next, these peptide-pulsed DCs were inactivated byX-ray irradiation (20 Gy), and mixed in a 1:20 ratio with autologousCD8⁺ T cells obtained by positive selection using the CD8 PositiveIsolation Kit (Dynal). These culture products were seeded in a 48-wellplate (Corning). Each well was made to contain 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8⁺ T cells and 10 ng/ml IL-7 (R&D System) in 0.5 ml of theAIM-V/2% AS medium. Three days later, these culture products were addedwith a final concentration of 20 IU/ml IL-2 (CHIRON). On day 7 and day14, T cells were further stimulated with peptide-pulsed autologous DCs.The DCs were prepared every time by the same method as above. On day 21,after the third peptide stimulation, CTLs were examined against thepeptide-pulsed C1R-A33 cell by a human interferon (IFN)-gammaenzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer2001, 84(1): 94-9; Umano Y et. al., Br J Cancer 2001, 84(8): 1052-7;Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al.,Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005,96(8): 498-506).

CTL Propagation Procedure

CTLs were propagated in culture using methods similar to those disclosedby Riddell et al. (Walter E A et al., N Engl J Med 1995, 333(16):1038-44; Riddell S R et al., Nat Med 1996, 2(2): 216-23). The CTLs wereco-cultured in a total of 25 ml AIM-V medium containing 5% AS (AIM-V/5%AS) and 40 ng/ml anti-CD3 antibody with two types of Mitomycin C-treatedhuman B lymphoblastoid cell lines at 5×10⁶ cells/flask. One day afterbeginning of the culturing, 120 IU/ml IL-2 was added to the culture. Ondays 5, 8 and 11, a fresh AIM-V/5% AS medium containing 30 IU/ml IL-2was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al.,Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006,97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

Establishment of CTL Clones

Dilution of CTLs was carried out to make 0.3, 1 and 3 cells/well in 96round-bottomed microtiter plates (Nalge Nunc International). The CTLswere co-cultured with two types of Mitomycin C-treated human Blymphoblastoid cell lines at 1×10⁴ cells/well in a total of 150micro-1/well AIM-V/5% AS medium with 30 ng/ml anti-CD3 antibody and 125IU/ml IL-2. Ten days later, IL-2 was added to the medium at 50micro-1/well to reach a final concentration of 125 IU/ml. On day 14, theCTL activity was tested, and the CTL clones were propagated using thesame method as described above (Uchida N et al., Clin Cancer Res 2004,10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; WatanabeT et al., Cancer Sci 2005, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, an IFN-gamma ELISPOT assay and anIFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed.Specifically, peptide-pulsed C1R-A33 (1×10⁴ cells/well) was prepared asthe stimulator cell. The induced CTLs, i.e., CTL lines and CTL clones,were used as the responder cell. The IFN-gamma ELISPOT assay andIFN-gamma ELISA were performed according to the manufacturer's manual.

Establishment of Target Cells Forcibly Expressing a Target Gene andHLA-A*3303

A cDNA encoding the open-reading frame of a target gene or HLA-A*3303was amplified by PCR. The PCR-amplified product was cloned into anexpression vector. Either or both of the target gene-expressing vectorand the HLA-A*3303-expressing vector were transfected into COST, whichis a cell line negative for the target gene and HLA, using Lipofectamine2000 (Invitrogen) following the manufacturer's recommended protocol. Twodays after transfection, the transfected cells were harvested usingversene (Invitrogen), and used as the target cell for CTL activity assay(5×10⁴ cells/well).

Results Prediction of KOC1-derived HLA-A*3303-binding peptides

Tables 2a and 2b show KOC1-derived 9mer peptides and 10 mer peptidesthat have been predicted to bind to HLA-A*3303 in the descending orderof binding affinity. A total of 37 peptides that potentially have anHLA-A*3303-binding ability was selected and investigated to determineepitope peptides.

TABLE 2a HLA-A33-binding 9mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 543 FYACQVAQR   98 61 517SSAEVVVPR  105  4 282 LAHNNFVGR  140 62 493 RVPSFAAGR  182 15 317ELTLYNPER  260 63 485 EVKLEAHIR  278 64 125 TYSSKDQAR  467 65 461EAQFKAQGR  555 66 286 NFVGRLIGK  933 67 225 TQSKIDVHR 1039 68  69EVEHSVPKR 1453 69 161 QQNPLQQPR 1485 70 406 ETVHLFIPA 2340 71  73SVPKRQRIR 2969 72 570 QSGPPQSRR 3001 73 121 VVNVTYSSK 3272  2 465KAQGRIYGK 3308  3 142 FQLENFTLK 4063  8  34 LVKTGYAFV 4107 74 211IGKEGATIR 4197 75 307 DTKITISPL 4676 76

TABLE 2b HLA-A33-binding 10mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 542 HFYACQVAQR   16 77 124VTYSSKDQAR  201 39 224 QTQSKIDVHR  296 42 281 ILAHNNFVGR  320 52  72HSVPKRQRIR  428 78 516 LSSAEVVVPR  467 35 496 SFAAGRVIGK 1710 34 543FYACQVAQRK 2635 79 424 QGQHIKQLSR 2752 80 190 KQKPCDLPLR 3492 81 431LSRFAGASIK 3726 37 568 ALQSGPPQSR 4478 45 210 IIGKEGATIR 4831 82 478NFVSPKEEVK 4831 83  77 RQRIRKLQIR 4883 84 285 NNFVGRLIGK 4883 85 Startposition indicates the number of amino acid residue from the N-terminusof KOC1. The dissociation constant [Kd (nM)] is derived from “NetMHCpan2.8”.Induction of CTLs by the Predicted KOC1-Derived HLA-A*3303-RestrictedPeptides

CTLs against the KOC1-derived peptides were generated according to theprotocol described in “Materials and methods”. The peptide-specific CTLactivity was measured by an IFN-gamma ELISPOT assay (FIG. 5). Incomparison with the control, CTLs in Well #1 with KOC1-A33-9-543 (SEQ IDNO: 61) (a), Well #2 with KOC1-A33-9-282 (SEQ ID NO: 62) (b), Well #3with KOC1-A33-9-317 (SEQ ID NO: 63) (c), Well #8 with KOC1-A33-9-485(SEQ ID NO: 64) (d), Well #3 with KOC1-A33-9-286 (SEQ ID NO: 67) (e),Well #8 with KOC1-A33-9-34 (SEQ ID NO: 74) (f), Well #4 withKOC1-A33-10-542 (SEQ ID NO: 77) (g), Well #3 with KOC1-A33-10-281 (SEQID NO: 52) (h), Well #6 with KOC1-A33-10-543 (SEQ ID NO: 79) (i), Well#1 with KOC1-A33-10-424 (SEQ ID NO: 80) (j), and Well #5 withKOC1-A33-10-285 (SEQ ID NO: 85) (k) showed potent IFN-gamma production.Meanwhile, despite that other peptides shown in Tables 2a and 2bpotentially have an HLA-A*3303-binding activity, specific CTL activitywas not detected as a result of stimulation by those peptides. Anexample of typical negative data is that specific IFN-gamma productionwas not observed from CTLs stimulated with KOC1-A33-9-517 (SEQ ID NO: 4)(l). As a result, eleven types of KOC1-derived peptides were selected aspeptides capable of inducing potent CTLs.

Establishment of CTL Lines and Clones Against the KOC1-DerivedHLA-A*3303-Restricted Peptides

CTL lines were established by propagating CTLs in Well #1 withKOC1-A33-9-543 (SEQ ID NO: 61) (a), Well #2 with KOC1-A33-9-282 (SEQ IDNO: 62) (b), Well #8 with KOC1-A33-9-485 (SEQ ID NO: 64) (c), Well #3with KOC1-A33-9-286 (SEQ ID NO: 67) (d), Well #4 with KOC1-A33-10-542(SEQ ID NO: 77) (e), and Well #3 with KOC1-A33-10-281 (SEQ ID NO: 52)(f), which showed peptide-specific CTL activity in the IFN-gamma ELISPOTassay. The CTL activity of these CTL lines was measured by IFN-gammaELISA (FIG. 6). These CTL lines showed potent IFN-gamma productionagainst target cells pulsed with the respective peptides, in comparisonwith target cells that have not been pulsed with the peptides. Further,CTL clones were established from the CTL lines by limiting dilution asdescribed in the “Materials and methods” section above, IFN-gammaproduction from the CTL clones against peptide-pulsed C1R-A33 wasmeasured by IFN-gamma ELISA. Potent IFN-gamma production was observed inCTL clones stimulated with KOC1-A33-9-543 (SEQ ID NO: 61) (a),KOC1-A33-9-282 (SEQ ID NO: 62) (b), KOC1-A33-9-485 (SEQ ID NO: 64) (c),KOC1-A33-9-286 (SEQ ID NO: 67) (d), KOC1-A33-10-542 (SEQ ID NO: 77) (e),and KOC1-A33-10-281 (SEQ ID NO: 52) (f) (FIG. 7).

Specific CTL Activity Against Target Cells Expressing KOC1 andHLA-A*3303

The CTL lines and CTL clones established against KOC1-A33-9-485 (SEQ IDNO: 64) (a), KOC1-A33-9-286 (SEQ ID NO: 67) (b), and KOC1-A33-10-542(SEQ ID NO: 77) (c) were investigated for their ability to recognizetarget cells expressing KOC1 and the HLA-A*3303 molecule. COS7 cellstransfected with both full-length KOC1 and the HLA-A*3303 gene (aspecific model of target cells expressing KOC1 and the HLA-A*3303 gene)were prepared as the target cell. COS7 cells transfected with eitherfull-length KOC1 or HLA-A*3303 were prepared as the control. The CTLclones stimulated with KOC1-A33-9-485 (SEQ ID NO: 64) (a),KOC1-A33-9-286 (SEQ ID NO: 67) (b), or KOC1-A33-10-542 (SEQ ID NO: 77)(c) demonstrated potent CTL activities against COS7 cells expressingboth KOC1 and HLA-A*3303 (FIG. 8). On the other hand, a significantspecific CTL activity was not detected against the control cells. Thesedata clearly proved that KOC1-A33-9-485 (SEQ ID NO: 64), KOC1-A33-9-286(SEQ ID NO: 67), and KOC1-A33-10-542 (SEQ ID NO: 77) are peptidesgenerated from endogenous processing of KOC1, and are presented ontarget cells with the HLA-A*3303 molecule and recognized by CTLs. Theseresults demonstrated the possibility that KOC1-A33-9-485 (SEQ ID NO:64), KOC1-A33-9-286 (SEQ ID NO: 67), and KOC1-A33-10-542 (SEQ ID NO: 77)may be suitable as a cancer vaccine for patients having aKOC1-expressing cancer.

Homology Analysis of Antigen Peptides

CTLs stimulated with KOC1-A33-9-543 (SEQ ID NO: 61), KOC1-A33-9-282 (SEQID NO: 62), KOC1-A33-9-317 (SEQ ID NO: 63), KOC1-A33-9-485 (SEQ ID NO:64), KOC1-A33-9-286 (SEQ ID NO: 67), KOC1-A33-9-34 (SEQ ID NO: 74),KOC1-A33-10-542 (SEQ ID NO: 77), KOC1-A33-10-281 (SEQ ID NO: 52),KOC1-A33-10-543 (SEQ ID NO: 79), KOC1-A33-10-424 (SEQ ID NO: 80), orKOC1-A33-10-285 (SEQ ID NO: 85) demonstrated significant specific CTLactivities. These results may be due to the fact that the KOC1-A33-9-543(SEQ ID NO: 61), KOC1-A33-9-282 (SEQ ID NO: 62), KOC1-A33-9-317 (SEQ IDNO: 63), KOC1-A33-9-485 (SEQ ID NO: 64), KOC1-A33-9-286 (SEQ ID NO: 67),KOC1-A33-9-34 (SEQ ID NO: 74), KOC1-A33-10-542 (SEQ ID NO: 77),KOC1-A33-10-281 (SEQ ID NO: 52), KOC1-A33-10-543 (SEQ ID NO: 79),KOC1-A33-10-424 (SEQ ID NO: 80), and KOC1-A33-10-285 (SEQ ID NO: 85)sequences are homologous to peptides derived from other molecules knownfor sensitizing the human immune system. In order to exclude thispossibility, homology analysis was performed by querying these peptidesequences using the BLAST algorithm (blast.ncbi.nlm.nih.gov/Blast.cgi).This result proved that the KOC1-A33-9-543 (SEQ ID NO: 61),KOC1-A33-9-317 (SEQ ID NO: 63), KOC1-A33-9-485 (SEQ ID NO: 64),KOC1-A33-9-34 (SEQ ID NO: 74), KOC1-A33-10-542 (SEQ ID NO: 77),KOC1-A33-10-543 (SEQ ID NO: 79), and KOC1-A33-10-424 (SEQ ID NO: 80)sequences are unique. On the other hand, KOC1-A33-9-282 (SEQ ID NO: 62),KOC1-A33-9-286 (SEQ ID NO: 67), KOC1-A33-10-281 (SEQ ID NO: 52), andKOC1-A33-10-285 (SEQ ID NO: 85) are identical to peptide sequencesidentified in IMP-1 which is another IMP family. KOC1-A33-9-485 (SEQ IDNO: 64) is identical to a peptide sequence identified in IMP-2. It hasbeen previously reported that IMP-1 is not expressed in normal organsexcept testis, fetal liver and placenta (Hammer N A et al.,Reproduction. 2005; 130(2): 203-12). It is reported to be associatedwith tumor progression in lung cancer patients (Kato T et al., ClinCancer Res. 2007; 13: 434-42). It has been previously reported thatIMP-2 is not expressed in normal organs except testis and fetal liver(Hammer N A et al., Reproduction. 2005; 130(2): 203-12). It is involvedin the proliferation of glioblastoma cancer stem cells (Janiszewska M etal., Genes Dev. 2012; 26(17): 1926-44). IMP-1 and IMP-2 are promising astarget antigens for cancer immunotherapy. Therefore, to the knowledge ofthe present inventors, there is almost no possibility that thesepeptides would elicit an unintended immune response against otherunrelated molecules. In conclusion, novel KOC1-derivedHLA-A33-restricted epitope peptides were identified. It was demonstratedthat the KOC1-derived epitope peptides are applicable for cancerimmunotherapy.

Example 3

Materials and Methods

Cell Lines

C1R, an HLA-A- and HLA-B-negative human B lymphoblastoid cell line, andCOST, an African green monkey kidney cell line, were purchased fromATCC.

Generation of Stimulator Cells with Stable HLA-A*0301 Expression

C1R (C1R-A03) that stably expresses HLA-A*0301 was used as a stimulatorcell. A cDNA encoding the open-reading frame of HLA-A*0301 was amplifiedby PCR and cloned into an expression vector. C1R cells were transfectedwith the expression vector, and then selected using G418 (Invitrogen)for two weeks. The G418-selected cells were seeded into wells containingG418-added culture medium in a 96-well plate, and further cultured for30 days. The exogenous HLA-A*0301 expression in C1R cells was verifiedby flow cytometric analysis.

Selection of Candidate KOC1-Derived Peptides

KOC1-derived 9mer and 10 mer peptides that bind to the HLA-A*0301molecule were predicted using the binding prediction server “NetMHC 3.2”(www.cbs.dtu.dk/services/NetMHC/) (Buus et al., Tissue Antigens. 2003November, 62(5): 378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17; Bioinformatics. 2004 Jun. 12: 20(9): 1388-97).

Peptide Synthesis

The peptides were synthesized by Biosynthesis (Lewisville, Tex.)according to a standard solid-phase synthesis method, and purified byreversed phase high-performance liquid chromatography (HPLC). Thepeptides were analyzed for their purity (>90%) and identity byanalytical HPLC and mass spectrometry, respectively. The peptides weredissolved in dimethylsulfoxide at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as theantigen-presenting cell to induce a cytotoxic T lymphocyte (CTL)response against peptides presented on human leukocyte antigens (HLAs).As described in the other sections, DCs were generated in vitro(Nakahara S et al., Cancer Res 2003, 63(14): 4112-8). Specifically,peripheral-blood mononuclear cells isolated from healthy volunteers(HLA-A*0301-positive) with the Ficoll-Paque plus solution (Pharmacia)were separated by attaching to plastic tissue culture dishes (BectonDickinson) and concentrated as a monocyte fraction. Themonocyte-concentrated population was cultured in the presence of 1000IU/ml granulocyte macrophage colony-stimulating factor (R&D System) and1000 IU/ml interleukin(IL)-4 (R&D System) in an AIM-V medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). Afterseven days of culturing, the cytokine-induced DCs were pulsed with 20micro-g/ml each of the synthesized peptides in an AIM-V medium at 37degrees C. for three hours in the presence of 3 micro-g/ml beta2-microglobulin. The generated cells appeared to express on their cellsurface DC-associated molecules such as CD80, CD83, CD86 and HLA classII (data not shown). Next, these peptide-pulsed DCs were inactivated byX-ray irradiation (20 Gy), and mixed in a 1:20 ratio with autologousCD8⁺ T cells obtained by positive selection using the CD8 PositiveIsolation Kit (Dynal). These culture products were seeded in a 48-wellplate (Corning). Each well was made to contain 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8⁺ T cells and 10 ng/ml IL-7 (R&D System) in 0.5 ml of theAIM-V/2% AS medium. Three days later, these culture products were addedwith a final concentration of 20 IU/ml IL-2 (CHIRON). On day 7 and day14, T cells were further stimulated with peptide-pulsed autologous DCs.The DCs were prepared every time by the same method as above. On day 21,after the third peptide stimulation, CTLs were examined against thepeptide-pulsed C1R-A03 by a human interferon (IFN)-gamma enzyme-linkedimmunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al.,Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006,97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

CTL Propagation Procedure

CTLs were propagated in culture using methods similar to those disclosedby Riddell et al. (Walter E A et al., N Engl J Med 1995, 333(16):1038-44; Riddell S R et al., Nat Med 1996, 2(2): 216-23). The CTLs wereco-cultured in a total of 25 ml AIM-V medium containing 5% AS (AIM-V/5%AS) and 40 ng/ml anti-CD3 antibody with two types of Mitomycin C-treatedhuman B lymphoblastoid cell lines at 5×10⁶ cells/flask. One day afterbeginning of the culturing, 120 IU/ml IL-2 was added to the culture. Ondays 5, 8 and 11, a fresh AIM-V/5% AS medium containing 30 IU/ml IL-2was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et al.,Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer Sci 2006,97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).

Establishment of CTL clones

Dilution of CTLs was carried out to make 0.3, 1 and 3 cells/well in 96round-bottomed microtiter plates (Nalge Nunc International). The CTLswere co-cultured with two types of Mitomycin C-treated human Blymphoblastoid cell lines at 1×10⁴ cells/well in a total of 150micro-1/well AIM-V/5% AS medium with 30 ng/ml anti-CD3 antibody and 125IU/ml IL-2. Ten days later, IL-2 was added to the medium at 50micro-1/well to reach a final concentration of 125 IU/ml. On day 14, theCTL activity was tested, and the CTL clones were propagated using thesame method as described above (Uchida N et al., Clin Cancer Res 2004,10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; WatanabeT et al., Cancer Sci 2005, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, an IFN-gamma ELISPOT assay and anIFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed.Specifically, peptide-pulsed C1R-A03 (1×10⁴ cells/well) was prepared asthe stimulator cell. The induced CTLs, i.e., CTL lines and CTL clones,were used as the responder cell. The IFN-gamma ELISPOT assay andIFN-gamma ELISA were performed according to the manufacturer's manual.

Establishment of Target Cells Forcibly Expressing a Target Gene andHLA-A*0301

A cDNA encoding the open-reading frame of a target gene or HLA-A*0301was amplified by PCR. The PCR-amplified product was cloned into anexpression vector. Either or both of the target gene-expressing vectorand the HLA-A*0301-expressing vector were transfected into COST, whichis a cell line negative for the target gene and HLA, using Lipofectamine2000 (Invitrogen) following the manufacturer's recommended protocol. Twodays after transfection, the transfected cells were harvested usingversene (Invitrogen), and used as the target cell for CTL activity assay(5×10⁴ cells/well).

Results

Prediction of KOC1-Derived HLA-A*0301-Binding Peptides

Tables 3a and 3b show KOC1-derived 9mer peptides and 10 mer peptidesthat have been predicted to bind to HLA-A*0301 in the descending orderof binding affinity. A total of 23 peptides that potentially have anHLA-A*0301-binding ability was selected and investigated to determineepitope peptides.

TABLE 3a HLA-A3-binding 9mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 121 VVNVTYSSK   84  2 258KSILEIMHK  124  1  52 KAIEALSGK  496  7  28 PVSGPFLVK  613  6 465KAQGRIYGK  650  3 142 FQLENFTLK  752  8 415 LSVGAIIGK  764  5 497FAAGRVIGK  927 11 559 QVKQHQQQK 1152 24

TABLE 3b HILA-A3-binding 10mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 120 AVVNVTYSSK   17 27 414ALSVGAIIGK   37 28 431 LSRFAGASIK   69 37 181 RQGSPGSVSK  104 38 456ITGPPEAQFK  211 29 204 TQFVGAIIGK  211 30 496 SFAAGRVIGK  215 34 552KIQEILTQVK  365 36 281 ILAHNNFVGR  459 52  73 SVPKRQRIRK  521 31 535VVVKITGHFY  682 41 420 IIGKQGQHIK  858 55 141 GFQLENFTLK 1112 44  14APSDLESIFK 1275 32 Start position indicates the number of amino acidresidue from the N-terminus of KOC1. The dissociation constant [Kd (nM)]is derived from “NetMHC3.2”.Induction of CTLs by the Predicted KOC1-Derived HLA-A*0301-RestrictedPeptides

CTLs against the KOC1-derived peptides were generated according to theprotocol described in “Materials and methods”. The peptide-specific CTLactivity was measured by an IFN-gamma ELISPOT assay (FIG. 9). Incomparison with the control, CTLs in Well #5 with KOC1-A03-10-120 (SEQID NO: 27) (a), Well #3 with KOC1-A03-10-204 (SEQ ID NO: 30) (b), andWell #5 with KOC1-A03-10-281 (SEQ ID NO: 52) (c) showed potent IFN-gammaproduction. Meanwhile, despite that other peptides shown in Tables 3aand 3b potentially have an HLA-A*0301-binding activity, specific CTLactivity was not detected as a result of stimulation by those peptides.An example of typical negative data is that specific IFN-gammaproduction was not observed from CTLs stimulated with KOC1-A03-10-414(SEQ ID NO: 28) (d). As a result, three types of KOC1-derived peptideswere selected as peptides capable of inducing potent CTLs.

Establishment of CTL Lines and Clones Against the KOC1-DerivedHLA-A*0301-Restricted Peptides

CTL line was established by propagating CTLs in Well #5 withKOC1-A03-10-120 (SEQ ID NO: 27), which showed peptide-specific CTLactivity in the IFN-gamma ELISPOT assay. The CTL activity of these CTLlines was measured by IFN-gamma ELISA (FIG. 10). These CTL lines showedpotent IFN-gamma production against target cells pulsed with therespective peptides, in comparison with target cells that have not beenpulsed with the peptides. Further, CTL clones were established from theCTL lines by limiting dilution as described in the “Materials andmethods” section above, IFN-gamma production from the CTL clones againstpeptide-pulsed C1R-A03 was measured by IFN-gamma ELISA. Potent IFN-gammaproduction was observed in CTL clone stimulated with KOC1-A03-10-120(SEQ ID NO: 27) (FIG. 11).

Specific CTL Activity Against Target Cells Expressing KOC1 andHLA-A*0301

The CTL clone established against KOC1-A03-10-120 (SEQ ID NO: 27) wasinvestigated for their ability to recognize target cells expressing KOC1and the HLA-A*0301 molecule. COS7 cells transfected with bothfull-length KOC1 and the HLA-A*0301 gene (a specific model of targetcells expressing KOC1 and the HLA-A*0301 gene) were prepared as thetarget cell. COS7 cells transfected with either full-length KOC1 orHLA-A*0301 were prepared as the control. The CTL clone stimulated withKOC1-A03-10-120 (SEQ ID NO: 27) demonstrated potent CTL activitiesagainst COS7 cells expressing both KOC1 and HLA-A*0301 (FIG. 12). On theother hand, a significant specific CTL activity was not detected againstthe control cells. These data clearly proved that KOC1-A03-10-120 (SEQID NO: 27) is a peptide generated from endogenous processing of KOC1,and is presented on target cells with the HLA-A*0301 molecule andrecognized by CTLs. These results demonstrated the possibility thatKOC1-A03-10-120 (SEQ ID NO: 27) may be suitable as a cancer vaccine forpatients having a KOC1-expressing cancer.

Homology Analysis of Antigen Peptides

CTLs stimulated with KOC1-A03-10-120 (SEQ ID NO: 27), KOC1-A03-10-204(SEQ ID NO: 30), or KOC1-A03-10-281 (SEQ ID NO: 52) demonstratedsignificant specific CTL activities. These results may be due to thefact that the KOC1-A03-10-120 (SEQ ID NO: 27), KOC1-A03-10-204 (SEQ IDNO: 30) and KOC1-A03-10-281 (SEQ ID NO: 52) sequences are homologous topeptides derived from other molecules known for sensitizing the humanimmune system. In order to exclude this possibility, homology analysiswas performed by querying these peptide sequences using the BLASTalgorithm (blast.ncbi.nlm.nih.gov/Blast.cgi). This result showed thatthere is no sequence having a significant homology with theKOC1-A03-10-120 (SEQ ID NO: 27) sequence. On the other hand,KOC1-A03-10-204 (SEQ ID NO: 30) is identical to a peptide sequenceidentified in IMP-2 which is another IMP family. KOC1-A03-10-281 (SEQ IDNO: 52) is identical to a peptide sequence identified in IMP-1 which isanother IMP family. It has been previously reported that IMP-1 is notexpressed in normal organs except testis, fetal liver and placenta(Hammer N A et al., Reproduction. 2005; 130(2): 203-12). It is reportedto be associated with tumor progression in lung cancer patients (Kato Tet al., Clin Cancer Res. 2007; 13: 434-42). It has been previouslyreported that IMP-2 is not expressed in normal organs except testis andfetal liver (Hammer N A et al., Reproduction. 2005; 130(2): 203-12). Itis involved in the proliferation of glioblastoma cancer stem cells(Janiszewska M et. al., Genes Dev. 2012; 26(17): 1926-44). IMP-1 andIMP-2 are promising as target antigens for cancer immunotherapy.Therefore, to the knowledge of the present inventors, there is almost nopossibility that these peptides would elicit an unintended immuneresponse against other unrelated molecules. In conclusion, novelKOC1-derived HLA-A03-restricted epitope peptides were identified. It wasdemonstrated that the KOC1-derived epitope peptides are applicable forcancer immunotherapy.

Example 4

Materials and Methods

Cell Lines

C1R, an HLA-A- and HLA-B-negative human B lymphoblastoid cell line, andCOST, an African green monkey kidney cell line, were purchased fromATCC.

Generation of Stimulator Cells with Stable HLA-A*0101 Expression

C1R (C1R-A01) that stably expresses HLA-A*0101 was used as a stimulatorcell. A cDNA encoding the open-reading frame of HLA-A*0101 was amplifiedby PCR and cloned into an expression vector. C1R cells were transfectedwith the expression vector, and then selected using G418 (Invitrogen)for two weeks. The G418-selected cells were seeded into wells containingG418-added culture medium in a 96-well plate, and further cultured for30 days. The exogenous HLA-A*0101 expression in C1R cells was verifiedby flow cytometric analysis.

Selection of Candidate KOC1-Derived Peptides

KOC1-derived 9mer and 10 mer peptides that bind to the HLA-A*0101molecule were predicted using the binding prediction server “NetMHC 3.2”(www.Cbs.Dtu.Dk/Services/NetMHC/) (Buus et al., Tissue Antigens. 2003November, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17; Bioinformatics. 2004 Jun. 12: 20(9): 1388-97).

Peptide Synthesis

The peptides were synthesized by American Peptide Company (Sunnyvale,Calif.) according to a standard solid-phase synthesis method, andpurified by reversed phase high-performance liquid chromatography(HPLC). The peptides were analyzed for their purity (>90%) and identityby analytical HPLC and mass spectrometry, respectively. The peptideswere dissolved in dimethylsulfoxide at 20 mg/ml and stored at −80degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as theantigen-presenting cell to induce a cytotoxic T lymphocyte (CTL)response against peptides presented on human leukocyte antigens (HLAs).As described in the other sections, DCs were generated in vitro(Nakahara S et al., Cancer Res 2003, 63(14): 4112-8). Specifically,peripheral-blood mononuclear cells isolated from healthy volunteers(HLA-A*0101-positive) with the Ficoll-Paque plus solution (Pharmacia)were separated by attaching to plastic tissue culture dishes (BectonDickinson) and concentrated as a monocyte fraction. Themonocyte-concentrated population was cultured in the presence of 1000IU/ml granulocyte macrophage colony-stimulating factor (R&D System) and1000 IU/ml interleukin(IL)-4 (R&D System) in an AIM-V medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). Afterseven days of culturing, the cytokine-induced DCs were pulsed with 20micro-g/ml each of the synthesized peptides in an AIM-V medium at 37degrees C. for three hours in the presence of 3 micro-g/ml beta2-microglobulin. Next, these peptide-pulsed DCs were inactivated byX-ray irradiation (20 Gy), and mixed in a 1:20 ratio with autologousCD8⁺ T cells obtained by positive selection using the CD8 PositiveIsolation Kit (Dynal). These culture products were seeded in a 48-wellplate (Corning). Each well was made to contain 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8⁺ T cells and 10 ng/ml IL-7 (R&D System) in 0.5 ml of theAIM-V/2% AS medium. Three days later, these culture products were addedwith a final concentration of 20 IU/ml IL-2 (CHIRON). On day 7 and day14, T cells were further stimulated with peptide-pulsed autologous DCs.The DCs were prepared every time by the same method as above. On day 21,after the third peptide stimulation, CTL activities were examinedagainst the peptide-pulsed C1R-A01 cells (Tanaka H et al., Br J Cancer2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7;Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al.,Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005,96(8): 498-506).

CTL Propagation Procedure

CTLs were propagated in culture using methods similar to those disclosedby Riddell et al. (Walter E A et al., N Engl J Med 1995, 333(16):1038-44; Riddell S R et al., Nat Med 1996, 2(2): 216-23). The CTLs wereco-cultured in a total of 25 ml AIM-V medium containing 5% AS and 40ng/ml anti-CD3 antibody with two types of Mitomycin C-treated human Blymphoblastoid cell lines at 5×10⁶ cells. One day after beginning of theculturing, 120 IU/ml IL-2 was added to the culture. On days 5, 8 and 11,a fresh AIM-V/5% AS medium containing 30 IU/ml IL-2 was added to theculture (Tanaka H et al., Br J Cancer 2001, 84(1): 94-9; Umano Y et al.,Br J Cancer 2001, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004,10(24): 8577-86; Suda T et al., Cancer Sci 2006, 97(5): 411-9; WatanabeT et al., Cancer Sci 2005, 96(8): 498-506).

Establishment of CTL Clones

CTLs were seeded to make 1 or 10 cells/well in 96 round-bottomedmicrotiter plates (Nalge Nunc International). The CTLs were co-culturedwith two types of Mitomycin C-treated human B lymphoblastoid cell linesat 1×10⁴ cells in a total of 150 micro-1/well 5% AS-containing AIM-Vmedium with 30 ng/ml anti-CD3 antibody and 125 IU/ml IL-2. Ten dayslater, 50 micro-1 of IL-2 was added to the medium to reach a final IL-2concentration of 125 IU/ml. On day 14, the CTL activity was tested, andthe CTL clones were propagated using the same method as described above(Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al.,Cancer Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005,96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, an IFN-gamma ELISPOT assay and anIFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed.Specifically, peptide-pulsed C1R-A01 (1×10⁴ cells/well) was prepared asthe stimulator cell. The induced CTLs, i.e., CTL lines and CTL clones,were used as the responder cell. The IFN-gamma ELISPOT assay andIFN-gamma ELISA were performed according to the manufacturer's manual.

Establishment of Target Cells Forcibly Expressing a Target Gene andHLA-A*0101

A cDNA encoding the open-reading frame of a target gene or HLA-A*0101was amplified by PCR. The PCR-amplified product was cloned into anexpression vector. Expression vectors were transfected usingLipofectamine 2000 (Invitrogen) following the manufacturer's recommendedprotocol. Two days after transfection, the transfected cells wereharvested using versene (Invitrogen), and used as the stimulator cellfor CTL activity assay (5×10⁴ cells/well).

Results

Prediction of KOC1-Derived HLA-A*0101-Binding Peptides

Tables 4a and 4b show KOC1-derived 9mer peptides and 10 mer peptidesthat have been predicted to bind to HLA-A*0101 in the descending orderof binding affinity. A total of 27 peptides that potentially have anHLA-A*0101-binding ability was selected and investigated to determineepitope peptides.

TABLE 4a HLA-A1-binding 9mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO  96 VLDSLLVQY    56 86 118ETAVVNVTY    57 87 114 NTDSETAVV   453 88 273 FTEEIPLKI   528 89 404ETETVHLFI  1528 90 536 VVKITGHFY  5589 13  15 PSDLESIFK  5882 12 143QLENFTLKV 11578 91 402 QSETETVHL 13101 92 305 DTDTKITIS 13406 93 313SPLQELTLY 21305 94 338 KAEEEIMKK 23866 10 301 KIEQDTDTK 24016 21  69EVEHSVPKR 24734 69

TABLE 4b HLA-A1-binding 10mer peptides  derived from KOC1 SEQ StartAmino acid Kd ID position sequence (nM) NO 312 ISPLQELTLY   146  46  95EVLDSLLVQY   498  50 402 QSETETVHLF  1737  95   9 LSENAAPSDL  2164  96535 VVVKITGHFY  3359  41 273 FTEEIPLKIL  4478  97 462 AQFKAQGRIY  6263 98 342 EIMKKIRESY  7665  99 252 GTSAACKSIL  9689 100  30 SGPFLVKTGY17794 101 114 NTDSETAVVN 22411 102 305 DTDTKITISP 29728 103  17DLESIFKDAK 35078 104 Start position indicates the number of amino acidresidue from the N-terminus of KOC1. The dissociation constant [Kd (nM)]is derived from “NetMHC3.2”.Induction of CTLs by the Predicted KOC1-Derived HLA-A*0101-RestrictedPeptides

CTLs against the KOC1-derived peptides were generated according to theprotocol described in “Materials and methods”. The peptide-specific CTLactivity was measured by an IFN-gamma ELISPOT assay (FIG. 13). Incomparison with the control, CTLs in Well #2 with KOC1-A01-9-96 (SEQ IDNO: 86) (a), Well #4 with KOC1-A01-9-118 (SEQ ID NO: 87) (b), Well #3with KOC1-A01-9-404 (SEQ ID NO: 90) (c), Well #4 with KOC1-A01-9-402(SEQ ID NO: 92) (d), Well #7 with KOC1-A01-10-312 (SEQ ID NO: 46) (e),Well #1 with KOC1-A01-10-402 (SEQ ID NO: 95) (f) and Well #1 withKOC1-A01-10-535 (SEQ ID NO: 41) (g) showed potent IFN-gamma production.Meanwhile, despite that other peptides shown in Tables 4a and 4bpotentially have an HLA-A*0101-binding activity, specific CTL activitywas not detected as a result of stimulation by those peptides. Anexample of typical negative data is that specific IFN-gamma productionwas not observed from CTLs stimulated with KOC1-A01-9-536 (SEQ ID NO:13) (h). As a result, seven types of KOC1-derived peptides were selectedas peptides capable of inducing potent CTLs.

Establishment of CTL Lines and Clones Against the KOC1-Derived Peptides

CTL lines were established by propagating CTLs in Well #2 withKOC1-A01-9-96 (SEQ ID NO: 86) (a) and Well #4 with KOC1-A01-9-118 (SEQID NO: 87) (b), which showed peptide-specific CTL activity in theIFN-gamma ELISPOT assay. The CTL activity of these CTL lines wasmeasured by IFN-gamma ELISA (FIG. 14). These CTL lines showed potentIFN-gamma production against target cells pulsed with the respectivepeptides, in comparison with target cells that have not been pulsed withthe peptides. Further, CTL clones were established from the CTL lines bylimiting dilution as described in the “Materials and methods” sectionabove, IFN-gamma production from the CTL clones against peptide-pulsedC1R-A01 cell was measured by IFN-gamma ELISA. Potent IFN-gammaproduction was observed in CTL clone stimulated with KOC1-A01-9-96 (SEQID NO: 86) (FIG. 15).

Specific CTL Activity Against Target Cells Expressing KOC1 andHLA-A*0101

The CTL clone established against KOC1-A01-9-96 (SEQ ID NO: 86) wasinvestigated for its ability to recognize target cells expressing KOC1and the HLA-A*0101 molecule. COS7 cells transfected with bothfull-length KOC1 and the HLA-A*0101 gene (a specific model of targetcells expressing KOC1 and the HLA-A*0101 gene) were prepared as thetarget cell. COS7 cells transfected with either full-length KOC1 orHLA-A*0101 were prepared as the control. The CTL clone stimulated withKOC1-A01-9-96 (SEQ ID NO: 86) demonstrated potent CTL activities againstCOS7 cells expressing both KOC1 and HLA-A*0101 (FIG. 16). On the otherhand, a significant specific CTL activity was not detected against thecontrol cells. These data clearly proved that KOC1-A01-9-96 (SEQ ID NO:86) is a peptide generated from endogenous processing of KOC1, and ispresented on target cells with the HLA-A*0101 molecule and recognized byCTLs. These results demonstrated the possibility that KOC1-A01-9-96 (SEQID NO: 86) may be suitable as a cancer vaccine for patients having aKOC1-expressing cancer.

Homology Analysis of Antigen Peptides

CTLs stimulated with KOC1-A01-9-96 (SEQ ID NO: 86), KOC1-A01-9-118 (SEQID NO: 87), KOC1-A01-9-404 (SEQ ID NO: 90), KOC1-A01-9-402 (SEQ ID NO:92), KOC1-A01-10-312 (SEQ ID NO: 46), KOC1-A01-10-402 (SEQ ID NO: 95),or KOC1-A01-10-535 (SEQ ID NO: 41) demonstrated significant specific CTLactivities. These results may be due to the fact that the KOC1-A01-9-96(SEQ ID NO: 86), KOC1-A01-9-118 (SEQ ID NO: 87), KOC1-A01-9-404 (SEQ IDNO: 90), KOC1-A01-9-402 (SEQ ID NO: 92), KOC1-A01-10-312 (SEQ ID NO:46), KOC1-A01-10-402 (SEQ ID NO: 95), and KOC1-A01-10-535 (SEQ ID NO:41) sequences are homologous to peptides derived from other moleculesknown for sensitizing the human immune system. In order to exclude thispossibility, homology analysis was performed by querying these peptidesequences using the BLAST algorithm (blast.ncbi.nlm.nih.gov/Blast.cgi).This result showed that there is no sequence having a significanthomology with the KOC1-A01-9-96 (SEQ ID NO: 86), KOC1-A01-9-118 (SEQ IDNO: 87), KOC1-A01-9-404 (SEQ ID NO: 90), KOC1-A01-9-402 (SEQ ID NO: 92),KOC1-A01-10-312 (SEQ ID NO: 46), KOC1-A01-10-402 (SEQ ID NO: 95), andKOC1-A01-10-535 (SEQ ID NO: 41) sequences. On the other hand,KOC1-A01-9-118 (SEQ ID NO: 87) is identical to peptide sequencesidentified in IMP-1 and IMP-2 which are the other IMP families. It hasbeen previously reported that IMP-1 is not expressed in normal organsexcept testis, fetal liver and placenta (Hammer N A et al.,Reproduction. 2005; 130(2): 203-12). It is reported to be associatedwith tumor progression in lung cancer patients (Kato T et al., ClinCancer Res. 2007; 13: 434-42). It has been previously reported thatIMP-2 is not expressed in normal organs except testis and fetal liver(Hammer N A et al., Reproduction. 2005; 130(2): 203-12). It is involvedin the proliferation of glioblastoma cancer stem cells (Janiszewska M etal., Genes Dev. 2012; 26(17): 1926-44). IMP-1 and IMP-2 are promising astarget antigens for cancer immunotherapy. Therefore, to the knowledge ofthe present inventors, there is almost no possibility that thesepeptides would elicit an unintended immune response against otherunrelated molecules. In conclusion, novel KOC1-derivedHLA-A01-restricted epitope peptides were identified. It was demonstratedthat the KOC1-derived epitope peptides are applicable for cancerimmunotherapy.

Example 5

Preparation of Emulsion Formulations

A peptide was dissolved in an injection solvent or sterile physiologicalsaline to become 1.0 mg/ml to 10.0 mg/ml, and collected into a syringe.This was connected via a connector to a syringe filled with an IFA in anamount equivalent to an injection solvent or sterile physiologicalsaline, and mixed by alternately pushing the syringe plungers of the twoconnected syringes. After several minutes of mixing, completion of theemulsion was assessed by the drop test method. The drop test method canbe performed by dropping one drop of the mixed sample on water. Theemulsion is assessed as completed when the sample dropped on water doesnot immediately diffuse in water; and the emulsion is assessed asincompleted when the sample dropped on water diffuses right away inwater. When the emulsion is assessed as incompleted, further mixing iscarried out to complete the emulsion. The completed emulsion can beadministered to a cancer patient by subcutaneous injection. The cancerpatient subject to administration can be selected from patients affectedby bladder cancer, cervical cancer, cholangiocellular cancer, chronicmyeloid leukemia (CML), colon cancer, rectum cancer, esophagus cancer,diffuse gastric cancer, non-small-cell lung cancer, small-cell lungcancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head andneck cancer, soft tissue tumor, testis cancer or such.

Preparation of Freeze-Dried Formulations

A peptide was dissolved in an injection solvent to become 1.0 mg/ml to10.0 mg/ml, and sterilized by filtration. This was filled into asterilized vial, and half-capped with a sterilized rubber plug. Afterthis vial was freeze-dried, it was completely capped and seamed with analuminum cap to produce a freeze-dried formulation. When in use, aninjection solvent or sterile physiological saline was injected into thevial to re-dissolve the freeze-dried powder. The re-dissolved solutionin the vial was collected using a syringe, and the syringe was connectedvia a connector with a syringe filled with an IFA in an amountequivalent to the collected re-dissolved solution. The re-dissolvedsolution and IFA were mixed by alternately pushing the syringe plungersof the two connected syringes. After several minutes of mixing,completion of the emulsion was assessed by the drop test method. Thecompleted emulsion can be administered to a cancer patient bysubcutaneous injection. The cancer patient subject to administration canbe selected from patients affected by bladder cancer, cervical cancer,cholangiocellular cancer, chronic myeloid leukemia (CML), colon cancer,rectum cancer, esophagus cancer, diffuse gastric cancer, non-small-celllung cancer, small-cell lung cancer, lymphoma, osteosarcoma, ovariancancer, kidney cancer, head and neck cancer, soft tissue tumor, testiscancer or such.

INDUSTRIAL APPLICABILITY

The present invention provides KOC1-derived novel HLA-A11-restricted,HLA-A33-restricted, HLA-A03-restricted and HLA-A01-restricted epitopepeptides that induce a potent and specific anti-tumor immune responseand thus have applicability for a wide range of cancer types. Thepeptides, compositions, APCs, and CTLs in the present invention can beused as a peptide vaccine for cancer expressing KOC1, for example,bladder cancer, cervical cancer, cholangiocellular cancer, chronicmyeloid leukemia (CML), colon cancer, rectum cancer, esophagus cancer,diffuse gastric cancer, non-small-cell lung cancer, small-cell lungcancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head andneck cancer, soft tissue tumor, and testis cancer.

While the present invention is herein described in detail and withrespect to specific embodiments thereof, it is to be understood that theforegoing description is exemplary and explanatory in nature and isintended to illustrate the present invention and its preferredembodiments. Through routine experimentation, one skilled in the artwill readily recognize that various changes and modifications can bemade therein without departing from the spirit and scope of the presentinvention, the metes and bounds of which are defined by the appendedclaims.

The invention claimed is:
 1. A composition comprising a pharmaceuticallyacceptable carrier and a peptide consisting of the amino acid sequenceselected from among SEQ ID NOs: 5, 28, 30, 32, 52, 27, 46 and 41; incombination with an adjuvant in an amount effective to enhance an immuneresponse.
 2. An emulsion comprising a water-soluble carrier; an oiladjuvant in an amount effective to enhance an immune response; and apeptide consisting of the amino acid sequence selected from among SEQ IDNOs: 5, 28, 30, 32, 52, 27, 46 and
 41. 3. A kit comprising a containerthat houses a composition comprising a pharmaceutically acceptablecarrier and a peptide consisting of the amino acid sequence selectedfrom among SEQ ID NOs: 5, 28, 30, 32, 52, 27, 46 and 41; and a containerthat houses an adjuvant in an amount effective to enhance an immuneresponse.
 4. A method of inducing an APC(s) having CTL-inducing ability,the method comprising a step of contacting an APC(s) with a peptide invitro, ex vivo or in vivo, wherein the peptide consists of the aminoacid sequence selected from among SEQ ID NOs: 5, 28, 30, 32, 52, 27, 46and
 41. 5. A method of inducing a CTL(s), the method comprising a stepof co-culturing a CD8-positive T cell(s) with an APC(s) that presents onits surface a complex of an HLA antigen and a peptide consisting of theamino acid sequence selected from among SEQ ID NOs: 5, 28, 30, 32, 52,27, 46 and
 41. 6. A method of inducing an immune response againstcancer, the method comprising administering to a subject a peptideconsisting of the amino acid sequence selected from among SEQ ID NOs: 5,28, 30, 32, 52, 27, 46 and
 41. 7. A method of treating cancer and/orpreventing postoperative recurrence thereof, the method comprisingadministering to a subject a peptide consisting of the amino acidsequence selected from among SEQ ID NOs: 5, 28, 30, 32, 52, 27, 46 and41.